TW202330612A - Compositions targeting bcma and methods of use thereof - Google Patents

Abstract

This present invention relates to BCMA binders (e.g. antibodies) and chimeric antigen receptor (CAR) constructs comprising a BCMA antigen binding molecule. The BCMA binders specifically bind to BCMA. The present BCMA CARs further comprise a hinge region (e.g., CD28 hinge), a transmembrane domain, and one or more intracellular NK cell signalling domains. NK cells expressing a BCMA CAR has increased efficacy in killing cancer cells. Provided herein also include therapeutic uses of the BCMA binders and BCMA CARs.

Description

Compositions targeting BCMA and methods of use Cross-references to related applications

This application claims the priority and rights of U.S. Provisional Application No. 63/257,822, filed on October 20, 2021; and U.S. Provisional Application No. 63/257,846, filed on October 20, 2021, each provisional application The contents are incorporated into this article by reference in full.

Sequence Listing Cross-Reference

This application is filed with a sequence listing submitted electronically in XML format. The sequence listing file is named MIL-019WO1_SL.XML, was created on October 12, 2022, and is 102,326 bytes in size; the electronic format information of the sequence listing is incorporated into this article by reference in its entirety.

The present application relates to BCMA conjugates (eg, antibodies) and chimeric antigen receptor (CAR) constructs comprising BCMA antigen-binding molecules. This application also includes therapeutic uses of BCMA conjugates and BCMA CAR.

B cell maturation antigen (also known as BCMA, CD269, TNFRSF17) is a non-glycosylated type I transmembrane protein, a member of the tumor necrosis receptor superfamily, and is preferentially expressed on differentiated plasma cells. now. BCMA has been shown to be overexpressed in various cancers, particularly various B cell-related cancers, including lymphoma, multiple myeloma, and others. Targeting BCMA has emerged as a promising approach to treat BCMA-positive cancers, including lymphoma, multiple myeloma, and other cancers. The most common treatments used to target BCMA include BCMA-specific antibodies (e.g., bispecific antibody constructs including BiTE® (bispecific T-cell engager) immuno-oncology therapies), antibody-drug conjugates (ADCs) ) and chimeric antigen receptor (CAR)-modified immune cell therapy.

Chimeric antigen receptor (CAR) technology has been designed not only to alleviate the generally immunosuppressive tumor microenvironment but also to redirect immune effector cells to cell surface tumor-specific antigens. CAR is artificially produced and used as a transmembrane receptor on immune effector cells to identify tumor cell surface antigens.

Natural killer (NK) cell lines are attractive contenders for CAR engineering because they mediate potent cytotoxicity against tumor cells and, unlike T cells, lack the ability to cause graft-versus-host disease (GVHD) in the allogeneic environment. ) potential. Therefore, NK cells can be used clinically as off-the-shelf cell therapy products. CAR-NK cells also retain the intrinsic ability to recognize and target tumor cells via their natural receptors, so in principle, the possibility of disease escape via down-regulation of CAR target antigens is smaller than that observed in the case of CAR-T cells .

The present disclosure provides novel anti-BCMA antibodies and B cell maturation antigen (BCMA) targeting CAR-NK cells, which can target the BCMA antigen on cancer cells through optimized CAR configurations.

The invention provides novel human anti-BCMA antibodies, antigen-binding fragments thereof, and In particular, novel BCMACAR constructs containing novel BCMA binders and NK cells expressing BCMA-CAR are provided. The present invention also provides methods of using novel BCMA conjugates, BCMA CARs, and/or BCMA-CAR expressing NK cells for the treatment of cancers associated with BCMA abnormalities. The present invention relates to antibodies and antigen-binding fragments thereof, as well as polynucleotides encoding human anti-BCMA antibodies, and chimeric antigen receptors (CARs) containing the anti-BCMA antibodies or antigen-binding fragments thereof of the invention that specifically bind BCMA. Specifically, the present invention is based on the observation that targeting CD28 containing a CAR-containing hinge domain compared to CAR-NK cells expressing an equivalent CAR construct except for having a different (e.g., IgG1) hinge domain. BCMA-based CAR-NK cells were unexpectedly more effective in tumor suppression in mouse multiple myeloma models. Accordingly, the present invention provides modified BCMA-CAR constructs for signaling in NK cells, resulting in more effective immunotherapy against BCMA-positive tumors.

In one aspect of the invention, provided herein include human anti-BCMA antibodies and antigen-binding fragments thereof; such human anti-BCMA antibodies and fragments thereof bind human BCMA with high specificity and affinity.

In some embodiments, the anti-BCMA antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region that includes three heavy chain complementary regions (HCDR), wherein HCDR1 includes SYAIH (SEQ ID NO: 2), HCDR2 contains VTWHDGSNKYYAESVMG (SEQ ID NO:3), and HCDR3 contains AKFGEPQYFQH (SEQ ID NO:4).

In some embodiments, an anti-BCMA antibody or antigen-binding fragment thereof of the invention binds BCMA with a _K greater than 0 and less than 150 nM. In some embodiments, an anti-BCMA antibody or antigen-binding fragment thereof binds BCMA with _{a K} greater than 1 pM and less than 10 nM. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof binds BCMA presented on human cells with an EC50 between 0.05 μg/ml and 0.5 μg/ml.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three light chain variable regions (LCDR), wherein LCDR2 comprises AASTLQS (SEQ ID NO: 7).

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three light chain variable regions (LCDRs), wherein LCDR1 comprises RASQGISSYLA (SEQ ID NO: 11), LCDR2 comprises AASTLQS (SEQ ID NO: 7), and LCDR3 contains QQLNSYPWT (SEQ ID NO: 14).

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three light chain variable regions (LCDRs), wherein LCDR1 comprises RASQGINNYLA (SEQ ID NO:6), LCDR2 comprises AASTLQS (SEQ ID NO:7), and LCDR3 contains QQLKSYPFT (SEQ ID NO: 8).

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three light chain variable regions (LCDRs), wherein LCDR1 comprises RASQGISSYLA (SEQ ID NO: 11), LCDR2 comprises AASTLQS (SEQ ID NO: 7), and LCDR3 contains QQLNSYPFT (SEQ ID NO: 12).

In some embodiments, the BCMA antibody or antigen-binding fragment thereof is an antibody comprising an IgG constant region.

In one embodiment, the present disclosure encompasses an anti-BCMA antibody, or antigen-binding fragment thereof, comprising three LCDRs, wherein,

LCDR1 contains RASQGIX ₁ X ₂ YLA (SEQ ID NO: 79),

LCDR2 contains AASTLQS (SEQ ID NO: 7), and/or

LCDR3 contains QQLX ₃ SYPX ₄ T (SEQ ID NO: 80);

Among them, X ₁ is selected from S or N;

X ₂ is selected from S or N;

X ₃ is selected from N or K; and/or

X ₄ is selected from F or W.

In some embodiments, an anti-BCMA antibody or antigen-binding fragment thereof comprises a variable heavy chain region (VH) comprising SEQ ID NO: 1. In some embodiments, an anti-BCMA antibody or antigen-binding fragment thereof comprises a VH that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least identical to SEQ ID NO: 1 99% consistent. In some embodiments, the BCMA antibody or antigen-binding fragment thereof comprises a VH comprising SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9 consistent.

In some embodiments, an anti-BCMA antibody or antigen-binding fragment thereof comprises a variable light chain region (VL) comprising SEQ ID NO: 5, 10, or 13. In some embodiments, the BCMA antibody or antigen-binding fragment thereof comprises a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, 10, or 13 % consistent.

In some embodiments, the BCMA antigen-binding fragment is selected from the group consisting of: IgA antibody, IgG antibody, IgE antibody, IgM antibody, bi- or multispecific antibody, Fab fragment, Fab' fragment, F(ab')2 Fragment, Fd' fragment, Fd fragment, isolated CDR or group thereof; single chain variable fragment (scFv), polypeptide-Fc fusion, single domain antibody, camel antibody; masking antibody, small module immunopharmaceutical (Small Modular ImmunoPharmaceutical, "SMIPsTM"), single-chain, tandem diabodies, VHH, anti-carrier proteins, nanobodies, minibodies, BiTE, ankyrin repeat proteins, DARPIN, Avimer, DART, TCR-like antibodies, adenatin, Human ubiquitin, transmembrane antibody (Trans-body); Affibody, TrimerX, MicroProtein, Fynomer, Centyrin; and KALBITOR.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a single chain variable fragment (scFv). In some embodiments, the anti-BCMA antibody or antigen-binding fragment comprises a linker sequence. In some embodiments, the anti-BCMA antibody or antigen-binding fragment comprises a linker selected from SEQ ID NO: 15-18. In some embodiments, the anti-BCMA antibody or antigen-binding fragment includes a signal peptide. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises the scFv of SEQ ID NO: 85-87. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a scFv that is at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96% identical to SEQ ID Nos: 85-87 , at least 97%, at least 98%, or at least 99% consistent.

In some embodiments, the present disclosure encompasses pharmaceutical compositions comprising an anti-BCMA antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. As non-limiting examples, anti-BCMA antibodies or antigen-binding fragments thereof include:

A heavy chain variable region containing three heavy chain complementary regions (HCDR), wherein HCDR1 contains SYAIH (SEQ ID NO: 2), HCDR2 contains VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 contains AKFGEPQYFQH (SEQ ID NO: 4). In other examples, an anti-BCMA antibody or fragment thereof can comprise a light chain variable complementarity determining region (LCDR) 2 comprising AASTLQS (SEQ ID NO: 7).

As a non-limiting example, the anti-BCMA antibody or antigen-binding fragment thereof of the present invention includes three HCDRs, including HCDR1, which includes SYAIH (SEQ ID NO: 2), and HCDR2, which includes VTWHDGSNKYYAESVMG (SEQ ID NO: 3). ), and HCDR3 comprising AKFGEPQYFQH (SEQ ID NO: 4); and three LCDRs comprising LCDR1 comprising RASQGINNYLA (SEQ ID NO: 6), LCDR2 comprising AASTLQS (SEQ ID NO: 7 ), and LCDR3 including QQLKSYPFT (SEQ ID NO: 8).

In one embodiment, the anti-BCMA antibody or antigen-binding fragment thereof of the invention includes three HCDRs, including HCDR1, which includes SYAIH (SEQ ID NO: 2), and HCDR2, which includes VTWHDGSNKYYAESVMG (SEQ ID NO: 3). ), and HCDR3 comprising AKFGEPQYFQH (SEQ ID NO: 4); and three LCDRs comprising LCDR1 comprising RASQGISSYLA (SEQ ID NO: 11), LCDR2 comprising AASTLQS (SEQ ID NO: 7 ), and LCDR3 containing QQLNSYPWT (SEQ ID NO: 14).

In another embodiment, the anti-BCMA antibody or antigen-binding fragment thereof of the invention includes three HCDRs, including HCDR1, which includes SYAIH (SEQ ID NO: 2), and HCDR2, which includes VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 comprising AKFGEPQYFQH (SEQ ID NO: 4); and three LCDRs comprising LCDR1 comprising RASQGISSYLA (SEQ ID NO: 11), LCDR2 comprising AASTLQS (SEQ ID NO: 7), and LCDR3 comprising QQLNSYPFT (SEQ ID NO: 12).

In another aspect of the invention, provided are chimeric antigen receptors (CARs) comprising an extracellular BCMA binding domain, a CD28 hinge region, a transmembrane domain, and one or more intracellular cell signaling domains, Wherein the BCMA binding domain comprises an anti-BCMA antibody or an antigen-binding fragment thereof as described herein. Thus, a BCMA-binding CAR as described herein recognizes and specifically binds BCMA antigen.

In some embodiments, the invention provides polynucleotides encoding chimeric antigen receptors (CARs) comprising a BCMA antigen binding domain, a CD28 hinge region, a transmembrane domain, and one or more intracellular cell signaling domains . In some embodiments, a polynucleotide can comprise at least one modification.

In some embodiments, the polynucleotide comprises a sequence encoding a BCMA antigen binding domain, The BCMA antigen binding domain comprises: (a) heavy chain variable region complementarity determining region (HCDR) 1, the HCDR1 comprising SEQ ID NO: 2; (b) HCDR2, the HCDR2 comprising SEQ ID NO: 3; and (c) HCDR3 comprising SEQ ID NO:4.

In some embodiments, the polynucleotide comprises a sequence encoding a BCMA antigen binding domain comprising: (a) light chain variable region complementarity determining region (LCDR) 1 comprising SEQ ID NO: 6; (b) LCDR2 comprising SEQ ID NO:7; and (c) LCDR3 comprising SEQ ID NO:8. In some embodiments, the polynucleotide comprises a sequence encoding a BCMA antigen binding domain comprising: (a) LCDR1 comprising SEQ ID NO: 11; (b) LCDR2 comprising SEQ ID NO :7; and (c) LCDR3, which includes SEQ ID NO:12. In some embodiments, the polynucleotide comprises a sequence encoding a BCMA antigen binding domain comprising: (a) LCDR1 comprising SEQ ID NO: 11; (b) LCDR2 comprising SEQ ID NO :7; and (c) LCDR3, which includes SEQ ID NO:14.

In some embodiments, the polynucleotide encodes a BCMA antigen binding domain comprising at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least SEQ ID NO: 1 or 9. About 99% or about 100% identical amino acid sequences. In some embodiments, the polynucleotide encodes a BCMA antigen binding domain comprising at least about 95%, at least about 96%, at least about 97% of any of SEQ ID NO: 5, 10, or 13. , an amino acid sequence that is at least about 98%, at least about 99%, or about 100% identical.

In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab fragment, F(ab')2 fragment, Fv fragment, or single chain variable fragment (scFv). In some embodiments, the antigen binding domain comprises a scFv. In some embodiments, the VH and VL of the scFv are connected by a linker. In some embodiments, The linker contains about 50 amino acids to about 2 amino acids. In some embodiments, the linker comprises at least 75%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence.

In some embodiments, the polynucleotide encodes a BCMA-binding CAR at less than about 1×10 ⁻⁶ M, less than about 1×10 ⁻⁷ M, less than about 1×10 ⁻⁸ M, or less than about 1×10 ⁻⁹ M of K _D combined with BCMA.

In some embodiments, the polynucleotide comprises a sequence encoding a transmembrane domain. The transmembrane domain of the BCMA-binding CAR of the present invention is CD28, CD3ζ, CD3 ε, CD3 γ, CD3 δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D or any combination thereof. In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

In some embodiments, the polynucleotide comprises a sequence encoding a hinge region. The hinge domain of the BCMA-binding CAR of the present invention is the CD28 hinge region. As non-limiting examples, the hinge region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, An amino acid sequence that is at least about 99% or about 100% identical.

In some embodiments, the polynucleotide comprises a sequence encoding a costimulatory region. In some embodiments, the costimulatory region is the following signaling region: CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), Inducible T cell costimulator (ICOS), CD8 γ, CD3 δ, CD3 ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF1.4), NKG2C, Igα (CD79a), Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, interleukin receptor, integrin, signaling lymphocyte-activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll Ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8α, CD8β, 11.2 β, IL2R γ , IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD18, LFA- 1. ITGB7, NKG2D, TNFR2, TRANCE RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D ), CD69, SLAMF6 (NTB-A, Ly108), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD 19a, ligands that specifically bind to CD83 or other Any combination. In some embodiments, the costimulatory region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% of SEQ ID NO: 28 , an amino acid sequence that is at least about 99% or about 100% identical.

In some embodiments, the polynucleotide comprises a sequence encoding an activation domain. As a non-limiting example, the activation domain is the CD3ζ domain. In some embodiments, the activation domain comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 7, at least about 98%, at least About 99% or about 100% identical amino acid sequences.

In some embodiments, the polynucleotide further comprises a sequence encoding a suicide gene. In some embodiments, the polynucleotide comprises a suicide gene selected from: rituximab, iCaspase 9, herpes simplex virus-thymidine kinase (HSV-tk), and ganciclovir, acyclovir, or FIAU ;Oxidoreductase and cycloheximide; Cytosine deaminase and 5-fluorocytosine; Thymidine kinase thymidylate kinase (Tdk::Tmk). In some embodiments, the suicide gene is iCaspase9.

In some embodiments, the polynucleotide further comprises a sequence encoding an interleukin. exist In some embodiments, the interleukin is selected from IL-7, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the interleukin is IL-15. In some embodiments, the amino acid sequence of IL-15 comprises SEQ ID NO: 23.

In some embodiments, a BCMA binding CAR as described herein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about SEQ ID NO: 19-21 An amino acid sequence that is 97%, at least about 98%, at least about 99%, or about 100% identical.

In some embodiments, the present disclosure includes a vector comprising a polynucleotide as in any of the previous embodiments.

In some embodiments, the vector system is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

In some embodiments, the present disclosure provides cells expressing a BCMA-binding CAR as described herein. In some embodiments, a CAR-expressing cell comprises a polynucleotide encoding a BCMA-binding CAR as described herein. In other embodiments, the CAR-expressing cells comprise vectors as described herein.

In some embodiments, the cell line is an immune cell. In some embodiments, the immune cell line is NK cells, T cells, or tumor-infiltrating lymphocytes (TILs), iNKT cells, B cells, macrophages, dendritic cells, or mixtures thereof. In some embodiments, the present disclosure relates to a population of immune cells comprising an immune cell as in any of the above embodiments.

In some embodiments, the present disclosure relates to compositions comprising a polynucleotide as in any one of the above embodiments, a vector as in any one of the above embodiments, a vector as in any one of the above embodiments The CAR of the item or the cell of any one of the above embodiments.

As a non-limiting example, the present disclosure relates to NK cells comprising a polynucleotide encoding a CAR comprising: (a) an antigen-binding molecule that specifically binds BCMA, comprising a heavy chain A variable region (VH) that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1 or 9; and a light chain A variable region (VL) that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 5, 10, or 13; ( b) CD28 hinge region; (c) transmembrane domain; and (d) one or more intracellular cell signaling domains.

In some aspects, the present disclosure encompasses a polynucleotide encoding a BCMA-binding CAR comprising: (a) a CD28 hinge, (b) a transmembrane domain, (c) a costimulatory domain, and (d) an IL-15 cell Interleukin.

In one aspect, the present disclosure encompasses immune cells having a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR includes: (a) an antigen-binding domain; (b) a CD28 hinge; and (c) CD28 transmembrane domain. In some embodiments, the immune cells comprise a CAR that binds to BCMA expressed on tumor cells.

In another aspect of the invention, provided are methods including using anti-BCMA antibodies, antigen-binding fragments thereof, BCMA-binding CARs, vectors, cells and compositions as described herein.

In some embodiments, the present disclosure provides methods of treating cancer in an individual using an anti-BCMA antibody, an antigen-binding fragment thereof, a BCMA-binding CAR, a polynucleotide, a vector, and/or a cell expressing a BCMA-binding CAR; the method includes administering to the individual The step of administering a therapeutically effective amount of any of the antibodies, CARs, polynucleotides, vectors, cells and compositions discussed in this disclosure.

In some embodiments, the method further includes the step of providing an effective amount of additional therapy to the individual.

In some embodiments, additional therapies include surgery, radiation, gene therapy, immunotherapy, or hormonal therapy.

In some embodiments, by infusion, injection, intravenous, intraarterial, intraperitoneal, Administering cells containing polynucleotides to an individual intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, transcutaneously, subcutaneously, topically, by perfusion, in the tumor microenvironment, or combinations thereof, or Cells containing vectors.

In some embodiments, the cancer is an immune cell malignancy, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is selected from multiple myeloma, lymphoma, and/or leukemia.

It should be noted that the various components shown in these and other figures are for illustrative purposes only and should not be read into the scope of the claims unless expressly recited therein.

Figure 1 is a schematic diagram of an exemplary BCMA-CAR construct with IL-15 interleukin (eg, soluble IL-15) and the hinge region from CD28.

Figure 2 is an in vitro cytotoxicity analysis of NK cells transduced with BCMA CAR with IgG hinge and CD28 hinge compared with non-transduced (NT) cells.

Figure 3 is a graphical representation showing tumor progression in NK cells co-administered with BCMA-CAR with IgG hinge or CD28 hinge domains.

Figure 4 is a graphical representation of tumor progression in the presence of different co-administered NK cells expressing BCMA-CAR with CD28 hinge domain (1M or 3M).

Figure 5 is a diagram showing different co-administered NK cells of BCMA-CAR (1M) with CD28 hinge domain.

Figure 6 is a graph showing BCMA-CAR expressing NK cells administered at a concentration of 1M or 3M one day after MM1S tumor cell inoculation ("1-day delayed dosing"); non-transduced NK cells served as control (NT-NK) Show.

Figure 7 is a Kaplan Meier survival curve for mice administered BCMA CAR-containing NK cells at doses of 1 million or 3 million one day after MM1s tumor cell inoculation ("1-day delayed dosing").

Figure 8 is a slide depicting histopathological analysis of lungs from mice collected after in vivo treatment with 10 million NK cells containing BCMA-CAR.

Figure 9A is a graph showing IL-15 secretion in mouse plasma following a 1-day delayed dose. Figure 9B is a graph showing IL-15 secretion in mouse plasma following a 9-day delayed dose. Figure 9C is a graph showing the proliferation of CAR-NK cells in the blood of mice under a 9-day delayed dose. Figure 9D is a graph showing the proliferation of CAR-NK cells in the blood of mice under a 1-day delayed dose.

Figure 10A is a graph showing the killing of tumor cell lines after treatment with BCMA-CART cells containing CD28 hinge or IgG hinge at different effector: target (E:T) ratios compared to C11D5.3VLVH-Fc. Figure 10B is a graph showing % apoptotic protease + MM1s target cells as a marker of apoptosis after treatment with BCMA-CART cells containing CD28 hinge or IgG hinge at different E:T ratios. Figure 10C is a graph showing % mitochondrial damage of BCMA in MM1s tumor cells after treatment with BCMA-CART cells containing CD28 hinge or IgG hinge at different E:T ratios.

Figures 11A-11B are graphical representations of the in vitro cytotoxicity of NK cells containing BCMA28-1 and BCMA28-2 CAR in the presence or absence of 800 ng/mL soluble BCMA, respectively. Figure 11C is a graphical representation of the in vitro cytotoxicity of the C11D5.3Fc positive control in the presence or absence of 800 ng/mL soluble BCMA.

Figure 12 shows the use of BCMA CAR NK at a 10M dose, administered 9 days after tumor cell inoculation ("9-day delayed dose") (Fig. 12A) and 1 day after tumor cell inoculation ("1-day delayed dose"). ) (Figure 12B) and graph of body weight fluctuations of treated mice.

Figure 13 shows representative images of the survival of mice inoculated with luciferase-expressing MM tumor cells and then received one dose (10Mx1) or two doses (10Mx2) of BCMA28-2 expressing CAR-NK cells.

Figure 14 shows the in vitro killing activity of BCMA28-2 CAR constructs containing NK cells generated in 4 independent cord blood unit (CBU) donors against various tumor cell lines. Figure 14A shows BCMA28-2 constructs containing CAR-NK cells in MM1s (BCMA ^low ) cells compared to equivalent untransduced (UTD) NK cells in vitro in 4 independent CBU donors. Killing activity. Figure 14B shows in vitro killing of BCMA28-2 constructs containing CAR-NK cells in JJN3 (BCMA KO) cells compared to equivalent untransduced (UTD) NK cells in 4 independent donors. active. Figure 14C shows BCMA28-2 constructs containing CAR-NK cells in RPMI-8226 (BCMA ^high ) cells compared to equivalent untransduced (UTD) NK cells in 4 CBU independent donors. In vitro killing activity. Figure 14D shows BCMA28-2 constructs containing CAR-NK cells in JIN parent (BCMA ⁱⁿ ) cells compared to equivalent untransduced (UTD) NK cells in vivo in 4 CBU independent donors. External killing activity.

Figures 15A and 15B show the repeated killing anti-tumor activity of CAR NK cells expressing BCMA28-2 in an in vitro MM1S tumor model after multiple rounds of tumor cell addition.

Figure 16 shows the in vivo efficacy of BCMA28-2 constructs against RPMI-8226 tumors.

definition

In order to be consistent with long-standing patent law practice, when used in this specification with words including the scope of the claim, the word "a/an" means one/an or more than one/an ( i.e. , at least one species) grammatical object of the article. For example, "an element" means one element or more than one element. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be performed relative to any other method or composition described herein, and that different embodiments can be combined.

Throughout this specification, unless the context otherwise requires, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements. but does not exclude any other step or element or group of steps or elements. "Consisting of" means including and being limited to anything that follows the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or mandatory and that other elements may not be present. "Consisting essentially of" means including any of the elements listed after that phrase and is limited to other elements that do not interfere with or contribute to the activities or actions specified in the disclosure for the listed elements . Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present, depending on them Depends on whether it affects the activity or function of the listed elements.

Throughout this specification, references to “one embodiment”, “an embodiment”, “a specific embodiment”, “a related embodiment”, “a certain embodiment”, “another embodiment” or “a further embodiment” or Reference in combination means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the aforementioned phrases appearing in various places throughout the specification are not necessarily referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the terms "or" and "and/or" are used to describe multiple combinations or mutually exclusive components. For example, "x, y and/or z" may refer to "x" alone, "y" alone, "z" alone, "x, y and z", "(x and y) or z", "x or (y and z)" or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from embodiments.

Throughout this application, the term "about" is used according to its simple and ordinary meaning in the field of cellular and molecular biology to mean that a value includes the standard deviation of the error of the device or method used to determine the value.

Affinity : As used herein, the term "affinity" refers to a binding moiety (e.g., an antigen binding moiety (e.g., a variable domain described herein) and/or an Fc receptor binding moiety (e.g., an FcRn binding moiety described herein) ) and a target (e.g., an antigen (e.g., BCMA) and/or an FcR (e.g., FcRn)) and indicates the strength of the binding interaction. In some embodiments, the measure of affinity is expressed as the dissociation constant (K _D ). In some embodiments, the binding moiety has a high affinity for the target (e.g., a K _of less than about 10 ^-7 M, less than about 10 ^-8 M, or less than about 10 ^-9 M, less than about 10 ^-10 , less than about 10 ^{- 10} , less than about 10 ^-11 , less than about 10 ^-12 ). In some embodiments, the binding moiety has low affinity for the target (e.g., a _K greater than about 10 ^-7 M, greater than about 10 ^-6 M, greater than about 10 ^-5 M, or greater than about 10 ^-4 M ). In some embodiments, the binding moiety has high affinity for the target at a first pH, low affinity for the target at a second pH, and intermediate affinity for the target at a pH level between the first pH and the second pH. .

About or Approximately : As used herein, the terms "approximately" or "approximately" when applied to one or more values of interest refer to a value that is similar to the stated reference value. In certain embodiments, the term "about" or "approximately" means in either direction (greater or less than) the stated 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6 of the reference value A range of values within %, 5%, 4%, 3%, 2%, 1% or less.

Antibody : As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable region, such as an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab, F(ab') ₂ , Fd, Fv and dAb fragments) as well as intact antibodies, such as IgA, IgG, IgE, IgD, IgM (and Its subtype) intact immunoglobulin. The light chain of an immunoglobulin can be of the kappa or lambda type.

An antigen-binding fragment or an antibody fragment thereof refers to a portion of an intact antibody. Antigen-binding fragments or antibody fragments thereof refer to the portion of an intact antibody that binds to an antigen (eg, BCMA). The antigen-binding fragment may contain the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, antibody mimetics, scFv and single chain antibodies.

Binding moiety : As used herein, a "binding moiety" is any molecule or portion of a molecule that is capable of specifically binding a target, e.g., a target of interest (e.g., an antigen (e.g., BCMA) and/or an FcR ( For example, FcRn)). Binding moieties include, for example, antibodies, antigen-binding fragments thereof, Fc regions or Fc fragments thereof, antibody mimetics, peptides and aptamers.

BCMA : As used herein, the term "BCMA" refers to B cell maturation antigen. Human BCMA protein consists of 184 amino acids: 1-54: extracellular domain; 55-77: transmembrane domain; 78-184: cytoplasmic domain. The amino acid sequence of BCMA includes:

(SEQ ID NO：22)，細胞外結構域序列以下劃線表示。

(SEQ ID NO: 22), the extracellular domain sequence is underlined.

BCMA lacks signaling peptides and is similar to other receptors such as BAFF receptor, transmembrane activator, cyclophilin ligand interactor and calcium regulator (TACI). These receptors play a major role in the maturation and differentiation of B cells into plasma cells. Their ligands include BAFF and APRIL, which are increased in MM patients. BCMA is a cell surface receptor, also known as CD269 and tumor necrosis factor receptor superfamily member 17 (TNFRSF17), encoded by the TNFRSF17 gene. This receptor is mainly expressed on mature B lymphocytes and most cases of multiple myeloma (MM).

Complementarity determining region (CDR) : The "CDR" of the variable domain is the amino acid residues in the variable domain, which are based on the definition of Kabat, Chothia, Kabat and Chothia accumulation, AbM, contact and/or conformation Definition or any CDR determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The positions of the CDRs can also be identified as structural loop structures originally described by Chothia and others. See, for example, Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition", which is a compromise between Kabat and Chothia and derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®); or the "AbM definition" of CDRs based on observed antigen contacts. Contact Definition," in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as "conformational definition" of CDRs, the position of the CDR can be identified as the residues that contribute enthalpically to antigen binding. See, eg, Makabe et al., Journal of Biological Chemistry, 283: 1 156-1166, 2008. There are other CDR boundary definitions that may not strictly follow one of the above methods, but still overlap at least part of the Kabat CDRs, although they may be shortened or lengthened, or even entirely, based on predictions or experimental results for specific residues or groups of residues. None of the CDRs significantly affects antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR may be defined according to any of Kabat, Chothia, Extension, AbM, Contact and/or Conformational definitions.

Chimeric Antigen Receptor (CAR) : A chimeric molecule that includes an antigen-binding moiety (such as a BCMA antibody) and a signaling domain, such as that from a T cell receptor (eg, CD3ζ). Generally, a CAR consists of an antigen-binding part, a transmembrane domain, and an intracellular domain. The intracellular domain typically includes a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FceRIy. In some cases, the intracellular domain further includes at least one additional costimulatory domain such as the intracellular portion of CD28 and/or CD137.

Constant Region : As used herein, the term "constant region" refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. Constant regions may include any or all of the following immunoglobulin domains: CH1 domain, hinge region, CH2 domain, CH3 domain (derived from IgA, IgD, IgG, IgE or IgM), and CH4 domain (derived from IgE or IgM).

Engineered : As used herein, the term "engineered" as used herein refers to artificially produced entities, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, the engineered entities are synthetic and contain elements that do not occur naturally or assembled in the manner in which they are used in this disclosure.

Epitope : As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope may be, for example, derived from two or more non-contiguous regions (conformational, non-linear, discontinuous) of one or more polypeptides. or noncontiguous epitopes). In certain embodiments, the epitope bound by the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA analysis, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), Determined by array-based oligopeptide scanning analysis and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (eg, Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software known in the art such as Refmac and Phenix. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. See, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for descriptions of mutagenesis techniques, including alanine scanning mutagenesis. .

Fc Region : As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides", each "Fc polypeptide" comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. In some embodiments, an "Fc region" includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the N-termini of these domains Flexible hinge. For IgG, an "Fc polypeptide" includes the immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3 (Cγ3) and the lower part of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of Fc polypeptides may vary, human IgG heavy chain Fc polypeptides are generally defined as containing residues starting from T223 or C226 or P230 to their carboxyl terminus, where numbering is according to Kabat et al. (1991, NIH Publication 91-3242, EU Index in National Technical Information Services, Springfield, VA). For IgA, the Fc polypeptide includes the immunoglobulin domains Calpha2 (Cα2) and Calpha3 (Cα3) and the lower part of the hinge between Calpha1 (Cα1) and Cα2. The Fc region can be synthetic, recombinant or produced from natural sources such as IVIG.

Isolated : As used herein, the term "isolated" refers to a molecule or biological product or cellular material that is substantially free of other materials. In one aspect, the term "isolated" refers to nucleic acids such as DNA or RNA that are separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs, respectively, such as those found in natural sources. , or protein or polypeptide, or cell or organelle, or tissue or organ. The term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture media when produced by recombinant DNA technology, or that are substantially free of chemical precursors or other chemicals when chemically synthesized . Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur naturally as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to a polypeptide isolated from other cellular proteins, and is intended to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues separated from other cells or tissues, and is intended to encompass cultured and engineered cells and tissues.

Ka : As used _herein , " _Ka " refers to the rate of association of a particular binding moiety with a target to form a binding moiety/target complex.

Kd : As used herein, " _Kd " refers to _the dissociation rate of a specific binding moiety/target complex.

KD : As used _herein , " _KD " refers to the dissociation constant, obtained from the ratio of _Kd to _Ka (i.e., _Kd / _Ka ) and expressed as molar concentration (M). The K _D value can be determined using methods well established in the art, for example, by using surface plasmon resonance, or using a biosensor system such as the Biacore® system.

Natural Killer Cells : Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. NK cells play a role similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells, acting approximately 3 days after infection, and respond to tumor formation.

，考慮到間隙的數量及每個間隙之長度，需要引入該等以實現兩個序列之最佳比對。此外，在確定兩個胺基酸序列之間的序列一致性程度時，技術人員可考慮所謂的「保守」胺基酸取代，其通常可描述為一個胺基酸殘基經另一個具有相似化學結構並且對多肽之功能、活性或其他生物學特性幾乎沒有影響或基本沒有影響的胺基酸殘基取代的胺基酸取代。此類保守的胺基酸取代係本領域熟知的，例如來自WO 04/037999、GB-A-2 357 768、WO 98/49185、WO 00/46383及WO 01/09300；並且此類取代之(較佳)類型及/或組合可基於來自WO 04/037999以及WO 98/49185及來自其中引用的進一步參考文獻之相關教示選擇。 Percent Identity: As used herein, "percent identity" is a function of the number of identical positions shared by similar phraseological sequences between two sequences ( i.e. ,

, taking into account the number of gaps and the length of each gap, these need to be introduced to achieve the optimal alignment of the two sequences. Additionally, when determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which can generally be described as the substitution of one amino acid residue by another chemically similar Amino acid substitutions of amino acid residues that have little or no effect on the structure of the polypeptide and have little or no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and such substitutions are ( Preferred) types and/or combinations may be selected based on the relevant teachings from WO 04/037999 and WO 98/49185 and from the further references cited therein.

Prevention : As used herein, "prevent" and similar words such as "prevented", "preventing", etc. mean to prevent, inhibit a disease or disorder such as cancer, or reduce the occurrence or recurrence of a disease or disorder. possible method. Prevention also means delaying the onset or recurrence of a disease or disorder or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms and/or burden of a disease or disorder before its onset or recurrence.

Sample : As used herein, the term "sample" generally refers to a biological sample. Samples may be taken from tissues or cells of an individual. In some examples, the sample may comprise or originate from tissue biopsy, blood (eg, whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, discarded tissue. Samples may have been separated from the source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymph fluid, saliva, urine, feces, tears, sweat or mucosal excretions, and other body fluids separated from the primary source prior to collection. In some examples, the sample is separated from its primary source (cells, tissue, body fluids such as blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or otherwise enriched from its primary source. In some cases, the primary source is homogenized before further processing. Samples can be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or enriched for nucleic acid, or may be treated with RNase. Samples may contain intact, fragmented, or partially degraded tissue or cells.

Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment" or "scFv" refers to an immunoglobulin that is covalently linked to form a _VH :: _VL heterodimer (e.g., A fusion protein of the variable regions of the heavy chain (V _H ) and the light chain (V _L ) of mouse or human. The heavy chain (V _H ) and the light chain (V _L ) are connected directly or through a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of V _H to V The C terminal of _L , or the C terminal of V _H and the N terminal of V _L. Linkers are typically rich in glycine to increase flexibility and serine or threonine to increase solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014/087010, the contents of which are incorporated herein by reference in their entirety.

Individual : As used herein, the term "individual" generally refers to an individual who has a biological sample being processed or analyzed and who, in a particular case, has or is suspected of having cancer. The subject may be any organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates such as apes, monkeys, orangutans, or chimpanzees), rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The individual may be a patient, for example, suffering from or suspected of suffering from a disease (which may be referred to as a medical disorder), such as benign or malignant neoplasia or cancer. The individual may be undergoing or have undergone treatment. Individuals may be asymptomatic. The individual can be a healthy individual but wish to prevent cancer. At least in some contexts, the term "individual" is used interchangeably. As used herein, a "subject" or "individual" may or may not be housed in a medical facility and may be considered an outpatient of the medical facility. An individual may be receiving one or more medical compositions via the Internet. An individual may include a human or non-human animal of any age, and thus includes adults and adolescents (ie, children) and infants, and includes individuals in utero. The term does not imply a need for medical treatment; therefore, individuals may participate voluntarily or involuntarily in clinical experiments or in support of basic scientific research.

Target : As used herein, a "target" is any molecule specifically bound by the binding portion of an antibody or antigen-binding fragment thereof. In some embodiments, the target is an antigen described herein (eg, BCMA). In some embodiments, the target is an FcR (eg, FcRn). As used herein, the terms "first target" and "second target" refer to two molecules of different molecular species, rather than two molecules of the same molecular species. For example, in some embodiments, the first target is serum protein and the second target is FcRn.

Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic molecule (e.g., an anti-BCMA antibody described herein) that confers a therapeutic effect to a subject at a reasonable benefit/risk ratio applicable to any medical treatment. . The effect of a treatment may be objective (ie, measurable by some test or marker) or subjective (ie, the individual gives an indication of an effect or feels an effect). Specifically, a "therapeutically effective amount" refers to an amount of a therapeutic molecule or composition that is effective in treating, ameliorating, or preventing a particular disease or disorder, or that exhibits a detectable therapeutic or preventive effect, such as by ameliorating symptoms associated with the disease. symptoms, prevent or delay the onset of disease, and/or also reduce the severity or frequency of disease symptoms. The therapeutically effective amount can be administered in a dosage regimen that may contain multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and/or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration, depending on combination with other agents. In addition, the specific therapeutically effective amount (and/or unit dose) for any particular individual will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific agent used; the specific composition used; The age, weight, general health, sex, and diet of the individual; the timing of administration, route of administration, and/or rate of excretion or metabolism of the specific therapeutic molecule employed; the duration of treatment; and similar factors well known in the medical arts.

Treatment : As used herein, the terms "treatment" or "treating" include any beneficial or desired effect on the symptoms or pathology of a disease or pathological disorder, and may include effects on the disease or disorder being treated. For example, even a minimal reduction in one or more measurable markers of cancer). Treatment may include reducing or ameliorating symptoms of the disease or disorder, or delaying the progression of the disease or disorder. "Treatment" does not necessarily mean complete eradication or cure of a disease or disorder or its related symptoms. By way of non-limiting example, treatment may partially or completely alleviate, ameliorate, alleviate, inhibit, delay the onset of, reduce the severity of, and/or reduce one or more of a particular disease, disorder, and/or disorder (e.g., BCMA-positive cancer). Any administration of a therapeutic molecule (eg, anti-BCMA antibodies and BCMA-binding CARs described herein) affects the incidence of symptoms or characteristics. In some cases, treatment may be directed to individuals who show no signs of the relevant disease, condition, and/or disorder and/or to individuals who only show early signs of the disease, condition, and/or disorder. Alternatively or additionally, such treatment may be directed to individuals exhibiting established signs of one or more of the relevant disease, condition and/or disorder.

Tumor Antigen: As used herein, "tumor antigen" means a biological molecule with antigenicity, the expression of which causes cancer.

Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described by "using" patent scope language, such as "using" any compound, composition, or agent discussed herein to achieve or perform the treatment described, diagnostic, or physiological purpose or effect.

I. Antibodies

The present disclosure is based in part on the Discovery of engineered antibodies and their antigen-binding fragments. BCMA is a protein with a single transmembrane domain, a cytoplasmic C-terminus and an extracellular N-terminus. BCMA is preferentially expressed by mature B lymphocytes, and its overexpression and activation are associated with enhanced expression of genes critical for survival, growth, adhesion, osteoclast activation, angiogenesis, metastasis, and immunosuppression. There is evidence that BCMA is expressed in various hematological malignancies, suggesting that BCMA may play an important role as a biomarker or therapeutic target in these diseases.

The anti-BCMA antibodies described herein are designed to bind to BCMA. In certain embodiments, the anti-BCMA antibodies and fragments thereof disclosed herein bind to human BCMA. In certain embodiments, human BCMA comprises or consists of the amino acid sequence having Uniprot reference number: Q02223 (SEQ ID NO: 22) or a fragment thereof. SEQ ID NO:22 is provided below:

(SEQ ID NO：22)

( SEQ ID NO: 22 )

In certain embodiments, the anti-BCMA antibodies and antigen-binding fragments thereof described herein bind to the extracellular domain of BCMA. In certain embodiments, anti-BCMA antibodies and antigen-binding fragments thereof bind to the extracellular domain of human BCMA. In certain embodiments, the extracellular domain of human BCMA comprises or consists of amino acids 1 to 54 of SEQ ID NO: 22.

In certain embodiments, the BCMA protein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% of the amino acid sequence listed in SEQ ID NO: 22 or a fragment thereof. %, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical amino acid sequences or consisting of.

The anti-BCMA antibodies described herein can be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other immunobinding moieties known in the art, including, for example, Fab , Fab', Fab'2, Fab ₂ , Fab ₃ , F(ab') ₂ , Fd, Fv, Feb, scFv, SMIP, antibody, diabody, trivalent antibody, quadrivalent antibody, minibody (minibody) , maxibody, tandab, DVD, BiTe, TandAb or similar, or any combination thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art.

Antibodies can be immunoglobulin molecules with four polypeptide chains, such as two heavy (H) chains and two light (L) chains. A heavy chain may include a heavy chain variable domain and a heavy chain constant domain. Heavy chain constant domains may include CH1, hinge, CH2, CH3, and (in some cases) CH4 regions. Suitable heavy chain constant regions may be derived from any immunoglobulin (eg, IgA, IgG, or IgE). In some embodiments, suitable heavy chain constant regions may be derived from IgG1, IgG2, or IgG4. In specific embodiments, suitable heavy chain constant regions are derived from IgG1. A light chain may include a light chain variable domain and a light chain constant domain. The light chain constant domain may include a kappa light chain or a lambda light chain. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain can usually be further subdivided into regions with variability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions ( FR). Such heavy chain and light chain variable domains may each include three CDRs and four framework regions, arranged in the following order from the amine terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, where One or more can be engineered as described herein. The assignment of amino acids to each domain is based on the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987); Chothia et al. Nature 342:878-883 (1989). As used herein, CDRs are referred to each of the heavy chain (HCDR1, HCDR2, HCDR3) and the light chain (LCDR1, LCDR2, LCDR3).

Embodiments of the invention include antibodies comprising CDRs found in the vH and vL domains described herein using commonly known numbering systems such as the IMGT, Kabat and Chothia numbering systems to identify. Such numbering systems are well known in the art.

heavy chain variable region

In some embodiments, anti-BCMA antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, an anti-BCMA antibody comprises a heavy chain variable region (VH) complementarity determining region (CDR) sequence:

vHCDR1: SYAIH (SEQ ID NO: 2)

vHCDR2: VTWHDGSNKYYAESVMG (SEQ ID NO: 3)

vHCDR3: AKFGEPQYFQH (SEQ ID NO: 4)

In certain embodiments, CDRs are identified according to the Kabat numbering system.

In some embodiments, the heavy chain variable region (VH) comprises an amino acid sequence

(SEQ ID NO：1)。

( SEQ ID NO: 1 ).

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Heavy chain amino acid sequence with 98% or 99% sequence identity:

(SEQ ID NO：54)。

( SEQ ID NO:54 ).

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:54 , a heavy chain amino acid sequence with 97%, 98% or 99% sequence identity, and also includes one or more of the vH CDR1, vHCDR2 and/or vHCDR3 sequences described herein.

In some embodiments, the engineered antibody comprises a heavy chain variable region having an amino acid sequence consistent with SEQ ID NO:1. In certain embodiments, the VH comprises at least about 70%, 75%, 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) the amino acid sequence set forth in SEQ ID NO: 1 ) identical or homologous amino acid sequences. For example, VH includes about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, About 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100 %identical or homologous amino acid sequences. In some embodiments, the anti-BCMA antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 , 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 1.

In some embodiments, the anti-BCMA variable heavy chain region is encoded by a polynucleotide comprising the following nucleic acid sequence:

(SEQ ID NO：55)

( SEQ ID NO:55 )

In some embodiments, the anti-BCMA antibody heavy chain is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, identical to SEQ ID NO: 56. 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity:

(SEQ ID NO：56)

( SEQ ID NO:56 )

In some embodiments, the engineered antibody comprises a heavy chain encoded by a polynucleotide having a nucleic acid sequence identical to SEQ ID NO: 56. In some embodiments, an anti-BCMA antibody comprises a nucleic acid sequence encoding an antibody comprising no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 54.

In some embodiments, an anti-BCMA antibody is encoded by a polynucleotide comprising a nucleic acid sequence encoding at least 70%, 75%, 80%, 85%, 90%, 91%, 92% identical to SEQ ID NO: 9 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the heavy chain amino acid sequence; also includes the vHCDR1, vHCDR2 and/or vHCDR3 sequences described herein one or more.

In some embodiments, anti-BCMA antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, an anti-BCMA antibody comprises a heavy chain variable region (VH) complementarity determining region (CDR) sequence:

vHCDR1: SYAIH (SEQ ID NO: 2)

vHCDR2: VTWHDGSNKYYAESVMG (SEQ ID NO: 3)

vHCDR3: AKFGEPQYFQH (SEQ ID NO: 4)

In certain embodiments, CDRs are identified according to the Kabat numbering system.

In some embodiments, the variable region of the heavy chain comprises an amino acid sequence

(SEQ ID NO：9)。

( SEQ ID NO:9 ).

In some embodiments, an anti-BCMA antibody comprises a heavy chain comprising: Amino acid sequences that have at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity:

(SEQ ID NO：57)。

( SEQ ID NO: 57 ).

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 57 , a heavy chain amino acid sequence with 97%, 98% or 99% sequence identity, and also includes one or more of the vHCDR1, vHCDR2 and/or vHCDR3 sequences described herein.

In some embodiments, the engineered antibody comprises a heavy chain amino acid sequence consistent with SEQ ID NO: 57. In certain embodiments, the VH comprises at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, or At least about 95%) identical or homologous amino acid sequences. For example, VH includes about 70%, 75%, 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86% of the amino acid sequence listed in SEQ ID NO: 9 %, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, About 99% or about 100% identical or homologous amine groups acid sequence. In some embodiments, the anti-BCMA antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 , 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 9.

In some embodiments, the anti-BCMA variable heavy chain is encoded by a polynucleotide comprising the following nucleic acid sequence:

(SEQ ID NO：58)

( SEQ ID NO:58 )

In some embodiments, a polynucleotide encoding an anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 Heavy chain nucleic acid sequence with %, 97%, 98% or 99% sequence identity:

(SEQ ID NO：59)

( SEQ ID NO:59 )

In some embodiments, the engineered antibody comprises a heavy chain nucleic acid sequence consistent with SEQ ID NO:59. In some embodiments, an anti-BCMA antibody comprises a nucleic acid sequence encoding an antibody comprising no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 9.

In some embodiments, an anti-BCMA antibody is encoded by a polynucleotide comprising a nucleic acid sequence encoding at least 70%, 75%, 80%, 85%, 90%, 91%, 92 identical to SEQ ID NO: 59 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the heavy chain amino acid sequence; also includes the vHCDR1, vHCDR2 and/or vHCDR3 sequences described herein one or more.

As one skilled in the art will appreciate, any such heavy chain CDR sequence may be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, such as by molecular biology techniques. , such any other antibody sequences or domains include any framework regions, CDRs or constant domains, or portions thereof as disclosed herein or otherwise known in the art, as may be present in the disclosure herein or otherwise known in the art. Any form of antibody or antigen-binding fragment thereof otherwise known in the art.

In the various engineered antibodies described herein, the heavy chain constant domains can be of any class (or subclass). In the various engineered antibodies described herein, the heavy chain constant domain may include the amino acid sequence of any one or more of IgG, IgM, IgA, IgD, or IgE, including subclasses such as IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2. In various embodiments, the constant domains of the engineered antibodies described herein may comprise a mixture of two or more classes (or subclasses) of immunoglobulin heavy chain constant domains. For example, an anti-BCMA antibody may include a first portion of a constant domain having an immunoglobulin constant domain sequence selected from the group consisting of IgG, IgM, IgA, IgD, or IgE class constant domains and having a constant domain sequence different from the first portion and selected from the group consisting of IgG, IgM, IgA , the second part of the constant domain of an immunoglobulin constant domain sequence of an IgD or IgE class constant domain. In some cases, the constant domains of the anti-BCMA antibodies described herein may include a mixture of two or more subclasses of a particular class of constant domains, such as an immunoglobulin with a constant domain selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 subclasses. A first part of the constant domain of a globulin constant domain sequence and a second part of a constant domain having an immunoglobulin constant domain sequence that is different from the first and is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subclass constant domains. In some specific embodiments, the constant domain includes all or part of the IgG2 constant domain and all or part of the IgG4 constant domain.

In some cases, anti-BCMA antibodies include antibody constant regions, Fc regions, or Fc fragments that exhibit alterations with one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors) Binding (compared to reference constant region). In some embodiments In, an anti-BCMA antibody includes an antibody constant region, Fc region, or Fc fragment that exhibits reduced binding (compared to a reference constant region) to one or more Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, or FcγRIV). Compare). In some embodiments, an anti-BCMA antibody includes an antibody constant region, Fc region, or Fc fragment that exhibits increased binding to the FcRn receptor at serum pH and/or intracellular pH (compared to a reference constant region).

For example, an anti-BCMA antibody may include the constant region, Fc region, or Fc fragment of an IgG antibody engineered to include amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 ( The amino acid addition, deletion or substitution of one or more of the Kabat numbers (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, NIH)). Without wishing to be bound by theory, it is believed that one or more of these constant region, Fc region or Fc fragment amino acids mediate interactions with Fc receptors, such as FcRn. In some embodiments, one or more of these disclosed amino acids is modified by histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, lysine, Asparagine or glutamic acid substitution. In some embodiments, non-histidine residues are substituted with histidine residues. In some embodiments, histidine residues are substituted with non-histidine residues.

In some embodiments, an anti-BCMA antibody includes a constant region, Fc region, or Fc fragment of an IgG antibody having amino acid modifications at one or more of positions 308, 309, 311, 312, and 314. In some embodiments, the substitution at one or more of positions 308, 309, 311, 312, and 314 is with threonine, proline, serine, aspartic acid, and leucine, respectively. . In some embodiments, residues at one or more of positions 308, 309, and 311 are substituted with isoleucine, proline, and glutamic acid, respectively. In yet other embodiments, the residues at one or more of positions 308, 309, 311, 312, and 314 are modified by threonine, proline, serine, aspartic acid, and leucine, respectively. replace.

In some embodiments, anti-BCMA antibodies include those having at positions 251, 252, 254, Amino acid modifications at one or more of 255 and 256, more specifically, constant regions, Fc regions or Fc fragments of IgG antibodies having substitutions at one or more of these positions. In some embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with leucine, tyrosine, phenylalanine, serine, tryptophan, or threonine, and residue 254 Substituted by threonine or serine, residue 255 is substituted by leucine, glycine, isoleucine or arginine, and/or residue 256 is substituted by serine, phenylalanine, arginine, Glutamine, glutamic acid, aspartic acid, alanine, asparagine or threonine substitution. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine or leucine, residue 254 is substituted with threonine or serine, and/or residue 255 is substituted with spermine acid substitution. In yet other embodiments, residue 252 is substituted with phenylalanine and/or residue 256 is substituted with aspartic acid. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and/or residue 255 is substituted with arginine.

In some embodiments, anti-BCMA antibodies include amino acid modifications at one or more of positions 428, 433, 434, 435, and 436, and more specifically, at one or more of these positions The constant region, Fc region or Fc fragment of the substituted IgG antibody. In some embodiments, residue 428 is substituted with methionine, threonine, leucine, phenylalanine, or serine, and residue 433 is substituted with lysine, arginine, serine, isoleucine Acid, proline, glutamine or histidine acid substitution, residue 434 is substituted by phenylalanine, tyrosine or histidine acid, residue 435 is substituted by tyrosine acid, and/or residue 436 is substituted by histamine Acid, asparagine, arginine, threonine, lysine, methionine or threonine substitution. In some embodiments, residues at one or more positions 433, 434, 435, and 436 are substituted with lysine, phenylalanine, tyrosine, and histidine, respectively. In some embodiments, residue 428 is substituted with methionine and/or residue 434 is substituted with tyrosine.

In some embodiments, an anti-BCMA antibody includes a protein having at positions 385, 386, 387 and amino acid modifications at one or more of 389, more specifically, constant regions, Fc regions or Fc fragments of IgG antibodies having substitutions at one or more of these positions. In some embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, or alanine, and residue 386 is substituted with threonine, proline , aspartic acid, serine, lysine, arginine, isoleucine or methionine, residue 387 is replaced by arginine, histidine, serine, threonine, propylamine acid or proline substitution, and/or residue 389 is substituted with proline or serine. In some embodiments, residues at one or more of positions 385, 386, 387, and 389 are substituted with arginine, threonine, arginine, and proline, respectively. In some embodiments, residues at one or more of positions 385, 386, and 389 are substituted with aspartic acid, proline, and serine, respectively.

In some embodiments, an anti-BCMA antibody includes a constant region, Fc region, or Fc fragment of an IgG antibody having one or more of the following substitutions: leucine at residue 251, tyrosine at residue 252, or Leucine, threonine or serine at residue 254, arginine at residue 255, threonine at residue 308, proline at residue 309, silk at residue 311 Amino acids, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, residue Proline at residue 389, methionine at residue 428, lysine at residue 433, phenylalanine or tyrosine at residue 434, tyrosine at position 435 and/or position Tyrosine at 436. Additional amino acid substitutions that may be included in the constant region, Fc region, or Fc fragment include those described, for example, in U.S. Patent Nos. 6,277,375; 8,012,476; and 8,163,881.

In some embodiments, anti-BCMA antibodies described herein include heavy chain constant domains, including, for example, PCT Publication Nos. WO 94/28027 and WO 98/47531; and Xu et al. (2000) Cell Immunol 200: Ala-Ala mutations described in 16-26. Thus, in some embodiments, anti-BCMA antibodies with one or more mutations within the heavy chain constant region including Ala-Ala mutations have reduced efficacy. Effector function or no effector function. According to these embodiments, the constant region of an anti-BCMA antibody described herein may comprise a substitution of alanine at position 234 and/or a mutation of alanine at position 235 (EU numbering).

As will be understood by those skilled in the art, any such heavy chain constant region sequence may be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, such as by molecular biology techniques. In combination, such any other antibody sequences or domains include any framework regions, CDRs or constant domains, or portions thereof as disclosed herein or otherwise known in the art, as may be present herein or otherwise known in the art. Any form of antibody or antigen-binding fragment thereof otherwise known in the art.

light chain variable region

Also provided are BCMA antibodies or fragments thereof that comprise various specific sequences in one or more light chain variable regions, including within the light chain complementarity determining regions LCDR1-3. In various embodiments, molecules having a particular light chain variable region are provided with heavy chain sequences as described above. In certain embodiments, CDRs are identified according to the Kabat numbering system.

Therefore, in some aspects, the invention provides anti-BCMA antibodies or antigen-binding fragments thereof, comprising RASQGISSYLA (SEQ ID NO: 11) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2) and QQLNSYPWT ( The light chain variable region of the complementarity determining region (CDR) sequence of SEQ ID NO: 14) (LCDR3).

In some embodiments, the invention provides anti-BCMA antibodies or antigen-binding fragments thereof, comprising RASQGINNYLA (SEQ ID NO: 6) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2) and QQLKSYPFT (SEQ ID NO: 8) The light chain variable region of the complementarity determining region (CDR) sequence of (LCDR3).

In some embodiments, the invention provides anti-BCMA antibodies or antigen-binding fragments thereof, comprising RASQGISSYLA (SEQ ID NO: 11) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2) and QQLNSYPFT (SEQ ID NO: 12) The light chain variable region of the complementarity determining region (CDR) sequence of (LCDR3).

In some embodiments, the invention provides anti-BCMA antibodies or antigen-binding fragments thereof comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 5, 10 or 13.

In some examples, the invention provides BCMA antibodies or fragments thereof comprising SYAIH (SEQ ID NO: 2) (HCDR1), VTWHDGSNKYYAESVMG (SEQ ID NO: 3) (HCDR2), and AKFGEPQYFQH (SEQ ID NO: 4) ( HCDR3) heavy chain variable complementarity determining region (CDR) sequence; and has RASQGISSYLA (SEQ ID NO: 11) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2) and QQLNSYPWT (SEQ ID NO: 14) ( The light chain variable region of the complementarity determining region (CDR) sequence of LCDR3).

In some embodiments, BCMA antibodies or fragments thereof comprise the heavy chains of SYAIH (SEQ ID NO: 2) (HCDR1), VTWHDGSNKYYAESVMG (SEQ ID NO: 3) (HCDR2), and AKFGEPQYFQH (SEQ ID NO: 4) (HCDR3) Variable complementarity determining region (CDR) sequences; and complementarity determinations with RASQGINNYLA (SEQ ID NO: 6) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2), and QQLKSYPFT (SEQ ID NO: 8) (LCDR3) Light chain variable region (CDR) sequence.

In some embodiments, BCMA antibodies or fragments thereof comprise the heavy chains of SYAIH (SEQ ID NO: 2) (HCDR1), VTWHDGSNKYYAESVMG (SEQ ID NO: 3) (HCDR2), and AKFGEPQYFQH (SEQ ID NO: 4) (HCDR3) Variable complementarity determining region (CDR) sequence; and having RASQGISSYLA (SEQ ID NO: 11) (LCDR1), AASTLQS (SEQ ID NO: 7) (LCDR2) and the light chain variable region of the complementarity determining region (CDR) sequence of QQLNSYPFT (SEQ ID NO: 12) (LCDR3).

In some embodiments, the BCMA antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising at least 70%, 75%, 80%, Amino acid sequences that are 85%, 90%, 95%, or 99% identical; and an immunoglobulin heavy chain variable (VH) region that contains at least 70% of SEQ ID NO: 1 , 75%, 80%, 85%, 90%, 95%, 99% identical amino acid sequences.

In some embodiments, the BCMA antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising at least 70%, 75%, 80%, A nucleic acid sequence that is 85%, 90%, 95%, or 99% identical; and an immunoglobulin heavy chain variable (VH) region that includes at least 70%, 75% of SEQ ID NO: 9 %, 80%, 85%, 90%, 95%, 99% identical nucleic acid sequences.

In some embodiments, the BCMA antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising at least 70%, 75%, 80%, Amino acid sequences that are 85%, 90%, 95%, or 99% identical; and an immunoglobulin heavy chain variable (VH) region that contains at least 70% of SEQ ID NO: 1 , 75%, 80%, 85%, 90%, 95%, 99% identical amino acid sequences.

In some embodiments, an anti-BCMA antibody or fragment thereof comprises a light chain variable region (VL) and/or LCDR having the amino acid sequence shown in Table 2 .

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 Variable light chain amino acid sequences with %, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, an anti-BCMA antibody comprises an antibody that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5, 10, or 13. The variable light chain amino acid sequence also includes one or more of the vLCDR1, vLCDR2 and/or vLCDR3 sequences described herein.

In some embodiments, an anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% sequence identity of the light chain amino acid sequence.

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:73 , light chain amino acid sequence with 97%, 98% or 99% sequence identity:

(SEQ ID NO：73)。

( SEQ ID NO:73 ).

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:74 , light chain amino acid sequence with 97%, 98% or 99% sequence identity:

(SEQ ID NO：74)。

( SEQ ID NO:74 ).

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:75 , light chain amino acid sequence with 97%, 98% or 99% sequence identity:

(SEQ ID NO：75)

( SEQ ID NO:75 )

In some embodiments, an anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% sequence identity of the light chain amino acid sequence, and also includes one or more of the vL CDR1, vLCDR2 and/or vLCDR3 sequences described herein.

In some embodiments, an anti-BCMA antibody or fragment thereof comprises a variable light chain (VL) amino acid sequence consistent with SEQ ID NO: 5, 10, and 13. In some embodiments, the anti-BCMA antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 , 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 5, 10 and 13.

In some embodiments, the anti-BCMA antibody comprises SEQ ID NO: 76-78. A light chain nucleic acid sequence of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity . In some embodiments, the engineered antibody comprises a light chain nucleic acid sequence consistent with SEQ ID NO: 76-78.

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:76 , light chain nucleic acid sequence with 97%, 98% or 99% sequence identity:

(SEQ ID NO：76)

( SEQ ID NO:76 )

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:77 , a light chain nucleic acid sequence with 97%, 98% or 99% sequence identity,

(SEQ ID NO：77)

( SEQ ID NO:77 )

In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO:78 , light chain nucleic acid sequence with 97%, 98% or 99% sequence identity:

(SEQ ID NO：78)

( SEQ ID NO:78 )

In some embodiments, an anti-BCMA antibody comprises a nucleic acid sequence encoding an antibody comprising no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NOs: 5, 10 and 13.

In some embodiments, the light chain of the anti-BCMA antibody or antigen-binding fragment thereof includes: three LCDRs, wherein,

LCDR1 contains RASQGIX ₁ X ₂ YLA (SEQ ID NO: 79),

LCDR2 contains AASTLQS (SEQ ID NO: 7), and/or

LCDR3 contains QQLX ₃ SYPX ₄ T (SEQ ID NO: 80);

Among them, X ₁ is selected from S or N;

X ₂ is selected from S or N;

X ₃ is a charged amino acid selected from N or K; and/or

X ₄ is a hydrophobic amino acid selected from F or W.

As one skilled in the art will appreciate, any such light chain CDR sequences may be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, such as by molecular biology techniques. , such any other antibody sequences or domains include any framework regions, CDRs or constant domains, or portions thereof as disclosed herein or otherwise known in the art, as may be present in the disclosure herein or otherwise known in the art. Any form of antibody or antigen-binding fragment thereof otherwise known in the art.

In some embodiments, the anti-BCMA antibodies described herein include a light chain including any light chain constant domain sequence, such as those known to those skilled in the art. As one skilled in the art will appreciate, the light chain constant domain may be a kappa light chain constant domain or a lambda light chain constant domain. In certain embodiments, a light chain constant domain as disclosed herein is a kappa light chain constant domain. In various embodiments, anti-BCMA antibodies described herein include a light chain constant domain.

Exemplary antibodies

Engineered antibodies can include various heavy and light chains described herein. In some embodiments, an anti-BCMA antibody can include two heavy and light chains. In various embodiments, the disclosure encompasses at least one heavy chain and/or light chain as disclosed herein, at least one heavy chain and/or light chain framework domain as disclosed herein, at least one heavy chain and/or light chain domain as disclosed herein, The disclosed heavy chain and/or light chain CDR domains, and/or as Antibodies to any heavy chain and/or light chain constant domain disclosed herein.

In some embodiments, the engineered antibody comprises an immunoglobulin VH amino acid sequence consistent with SEQ ID NO: 1 and an immunoglobulin VL amino acid sequence consistent with SEQ ID NO: 13. In some embodiments, a BCMA antibody or fragment thereof comprises an immunoglobulin VL region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13; and an immunoglobulin VH region comprising an amino acid sequence identical to SEQ ID NO: 13; 1 At least 90% identical amino acid sequence. In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, VH and/or VL amino acid sequences with 94%, 95%, 96%, 97%, 98% or 99% sequence identity, also including vH CDR1, vHCDR2, vHCDR3, vLCDR1, vLCDR2 and/or described herein or one or more of vLCDR3.

In some embodiments, anti-BCMA antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, anti-BCMA antibodies comprise vHCDR and vLCDR sequences:

vHCDR1: SYAIH (SEQ ID NO: 2)

vHCDR2: VTWHDGSNKYYAESVMG (SEQ ID NO: 3)

vHCDR3: AKFGEPQYFQH (SEQ ID NO: 4)

vLCDR1:RASQGISSYLA(SEQ ID NO:11)

vLCDR2:AASTLQS(SEQ ID NO:7)

vLCDR3: QQLNSYPWT (SEQ ID NO: 14)

In certain embodiments, CDRs are identified according to the Kabat numbering system.

In some embodiments, the engineered antibody comprises an immunoglobulin VH amino acid sequence consistent with SEQ ID NO: 1 and an immunoglobulin VL amino acid sequence consistent with SEQ ID NO:5. In some embodiments, a BCMA antibody or fragment thereof comprises an immunoglobulin VL region comprising the same sequence as SEQ ID NO: 5 is an amino acid sequence that is at least 90% identical; and an immunoglobulin VH region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, VH and/or VL amino acid sequences with 94%, 95%, 96%, 97%, 98% or 99% sequence identity, also including vH CDR1, vHCDR2, vHCDR3, vLCDR1, vLCDR2 and/or described herein or one or more of vLCDR3.

In some embodiments, anti-BCMA antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, anti-BCMA antibodies comprise vHCDR and vLCDR sequences:

vHCDR1: SYAIH (SEQ ID NO: 2)

vHCDR2: VTWHDGSNKYYAESVMG (SEQ ID NO: 3)

vHCDR3: AKFGEPQYFQH (SEQ ID NO: 4)

vLCDR1:RASQGINNYLA(SEQ ID NO:6)

vLCDR2:AASTLQS(SEQ ID NO:7)

vLCDR3: QQLKSYPFT (SEQ ID NO: 8)

In certain embodiments, CDRs are identified according to the Kabat numbering system.

In some embodiments, the engineered antibody comprises an immunoglobulin VH amino acid sequence consistent with SEQ ID NO:9 and an immunoglobulin VL amino acid sequence consistent with SEQ ID NO:10. In some embodiments, a BCMA antibody or fragment thereof comprises an immunoglobulin VL region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10; and an immunoglobulin VH region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10; 9. At least 90% identical amino acid sequence. In some embodiments, the anti-BCMA antibody comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, VH and/or VL amines with 94%, 95%, 96%, 97%, 98% or 99% sequence identity The amino acid sequence also includes one or more of vH CDR1, vHCDR2, vHCDR3, vLCDR1, vLCDR2 and/or vLCDR3 described herein.

In some embodiments, anti-BCMA antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, anti-BCMA antibodies comprise vHCDR and vLCDR sequences:

vHCDR1: SYAIH (SEQ ID NO: 2)

vHCDR2: VTWHDGSNKYYAESVMG (SEQ ID NO: 3)

vHCDR3: AKFGEPQYFQH (SEQ ID NO: 4)

vLCDR1:RASQGISSYLA(SEQ ID NO:11)

vLCDR2:AASTLQS(SEQ ID NO:7)

vLCDR3: QQLNSYPFT (SEQ ID NO: 12)

In certain embodiments, CDRs are identified according to the Kabat numbering system.

Exemplary single-chain variable fragments

In some embodiments, the present disclosure provides single-chain variable fragments. In some embodiments, the scFv is human scFv. "Single chain variable fragment" or "scFv" refers to the heavy ( _VH ) and light chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a _VH ::VL heterodimer. Fusion protein of the variable region of (V _L ). The heavy chain ( _VH ) and the light chain ( _VL ) are connected directly or through a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of _VH to V The C terminal of _L , or the C terminal of V _H and the N terminal of V _L. Linkers are typically rich in glycine to increase flexibility and serine or threonine to increase solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014/087010, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the linker is a G4S linker.

Alternatively or additionally, the scFv may be derived from a Fab' (rather than from an antibody, eg obtained from a Fab library). In certain embodiments, the anti-BCMA antibody or fragment thereof is a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the anti-BCMA antibody or fragment thereof is F(ab) ₂ . Any of the aforementioned molecules can be included in a fusion protein with heterologous sequences to form an anti-BCMA antigen antibody or antigen-binding fragment thereof.

In certain embodiments, the anti-BCMA antibody or fragment thereof is present at at least about 1x10 ^-12 M, at least about 1x10 ^-7 M, at least about 1x10 ^-8 M, at least about 1x10 ^-9 M, or at least about 1x10 ^-10 M. Dissociation constant (K _d ) for binding to BCMA (eg, human BCMA). In certain embodiments, an anti-BCMA antibody or fragment thereof binds to BCMA (eg, human BCMA) with a dissociation constant (K _D ) of at least about 2x10 ^-8 M. In certain embodiments, an anti-BCMA antibody or antigen-binding fragment thereof binds to BCMA (eg, human BCMA) with a dissociation constant ( _KD ) between about 2×10 ⁻⁸ M and about 8×10 ⁻⁹ M.

In some embodiments, an anti-BCMA antibody or fragment thereof binds to BCMA (e.g., human BCMA) with a dissociation constant ( _KD ) of about 1 nM to 50 nM, about 5 nM to 30 nM, about 5 nM to 25 nM, or about 8 nM to 20 nM. In some embodiments, the anti-BCMA antibody or fragment thereof is at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least A dissociation constant ( _KD ) binds to BCMA (e.g., human BCMA).

In some embodiments, the anti-BCMA scFv comprises a variable heavy chain comprising SEQ ID Nos: 1-4. In some embodiments, an anti-BCMA scFv comprises a variable heavy chain comprising one or more CDR sequences provided in Table 1. In some embodiments, the anti-BCMA scFv package Contains a variable light chain comprising one or more light chain sequences provided in Table 2.

In some embodiments, the anti-BCMA scFv comprises a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 provided below:

GGGGSGGGGSGGGGS ( SEQ ID NO: 15 ). In some embodiments, the anti-BCMA scFv comprises a linker comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 81 provided below:

ggagggggcggtagcggagggggaggatctgggggtgggggctcc ( SEQ ID NO: 81 )

In some embodiments, the linker comprises or consists of the amino acid sequence listed below:

GGGGSGGGGSGGGSGGGGS ( SEQ ID NO: 16 )

In some embodiments, the anti-BCMA scFv comprises a linker comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 82 provided below:

ggggggggggggagcggagggggggggagtggtggggggtcaggagggggaggaagt ( SEQ ID NO: 82 )

In some embodiments, the linker comprises or consists of the amino acid sequences listed below:

GGGGSGGGGSGGGGSGGGSGGGGS ( SEQ ID NO: 17 )

In some embodiments, the anti-BCMA scFv comprises a linker comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 83 provided below:

gggggggggggatcaggaggcggtgggagcgggggaggtggatccggtggagggtcaggaggtggagggtcc ( SEQ ID NO: 83 ).

In some embodiments, the linker comprises or consists of the amino acid sequences listed below:

GGGGSGGGGSGGGGSGGGGSGGGSGGGGS ( SEQ ID NO: 18 )

In some embodiments, the anti-BCMA scFv comprises a linker comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 84 provided below:

ggtggtggcggcagcggcggcggcggtagcggtggcggcggttctggaggaggaggcagcggtggaggaagcggaggtggaggctcc ( SEQ ID NO: 84 ).

In some embodiments, an anti-BCMA antibody or fragment thereof contains conservative sequence modifications (eg, an anti-BCMA antibody or fragment thereof described herein). In some embodiments, conservative sequence modifications are amino acid modifications that do not significantly affect or alter the binding characteristics of the presently disclosed anti-BCMA antibodies or fragments thereof (eg, antibodies or fragments thereof) comprising the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into anti-BCMA antibodies or fragments thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified based on their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified according to charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutral Charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, silk Amino acids, threonine, tryptophan, tyrosine and valine. In addition, amino acids can be classified according to their polarity: polar amino acids include arginine (alkaline polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamic acid , histamine (alkaline polarity), lysine (alkaline polarity), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isosine Leucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained functionality. In certain embodiments, no more than one, no more No more than two, no more than three, no more than four, no more than five residues are changed.

In some embodiments, the anti-BCMA scFv comprises the amino acid sequence:

(SEQ ID NO：85)

( SEQ ID NO:85 )

In some embodiments, the anti-BCMA scFv comprises the following amino acid sequence:

(SEQ ID NO：86)

( SEQ ID NO:86 )

In some embodiments, the anti-BCMA scFv comprises the following amino acid sequence:

(SEQ ID NO：87)

( SEQ ID NO: 87 )

In some embodiments, the anti-BCMA scFv comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 Amino acid sequences with %, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the anti-BCMA scFv comprises at least 70%, 75%, 80%, 85%, An amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and including the corresponding variable region. In some embodiments, the anti-BCMA scFv comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, and also includes the corresponding CDR region.

Nucleotide sequence

The disclosure includes encoding one or more heavy chains, heavy chain variable domains, heavy chain framework regions, heavy chain CDRs, heavy chain constant domains, light chains, light chain variable domains, light chain framework regions, light chain CDRs, Light chain constant domains, or other immunoglobulin-like sequences, or nucleotide sequences of the antibodies disclosed herein. In some embodiments, the nucleotide sequence is codon-optimized for mammalian expression. In various embodiments, such nucleotide sequences may be present in a vector. In various embodiments, such nucleotides may be present in the genome of cells, such as cells of an individual in need of treatment or cells used to produce antibodies, such as mammalian cells used to produce antibodies.

Engineered antibodies and fusion proteins

In some embodiments, the present disclosure provides fusion proteins comprising (i) one or more antigen-binding regions described herein (e.g., antigen-binding regions of immunoglobulins, heavy chain antibodies, light chain antibodies, based on Antibodies or other protein scaffolds with antibody-like properties for LRR, as well as other antigen-binding moieties known in the art, including, for example, Fab, Fab', _Fab'2 , Fab2, _Fab3 , F(ab') ₂ , Fd , Fv, Feb, scFv, SMIP, antibody, diabody, trivalent, quadrivalent, minibody, maximal antibody, tandab, DVD, BiTe, TandAb or the like), such as one or more of those described herein A variable domain, or a portion thereof (eg, one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, albumin is an abundant serum protein that is protected from degradation through pH-dependent cycling mediated by interaction with FcRn. In some embodiments, one or more variable domains or engineered antibodies as described herein, or portions thereof (e.g., one or more CDRs as described herein) are combined with albumin, a portion thereof (such as one that binds to FcRn). albumin moiety) and/or fused to an engineered variant of albumin that binds to FcRn with increased affinity. In other cases, one or more variable domains or engineered antibodies as described herein, or portions thereof (eg, one or more CDRs as described herein) are fused to a polypeptide that binds to albumin to form a fusion protein- Albumin complex, which in turn binds to FcRn. In some embodiments, the polypeptide that binds to albumin is a single chain variable fragment (scFv). Albumin, or portions thereof, may include mutations in one or more amino acids that alter its binding to FcRn. Such mutations are known in the art (see, eg, Andersen et al., Nature Communications 3:610 doi:10.1038/nocmms1607 (2012)). In other cases, one or more variable domains or engineered antibodies described herein, or portions thereof (eg, one or more CDRs described herein) are fused to transferrin. Transferrin is recycled by binding to the transferrin receptor (see, eg, Widera et al., Adv. Drug Deliv. Rev. 55:1439-66 (2003)).

Chimeric Antigen Receptor (CAR)

In some examples, BCMA antibodies can also be combined with antigen-specific such as tumor antigen-specific CARs (also known as chimeric antigen receptors, artificial T cell receptors, or chimeric immune receptors) and/or engineered to express CARs. used in combination with cytotoxic T lymphocytes (CTL). Typically, a CAR includes a binding moiety, extracellular hinge and spacer elements, a transmembrane region, and an intracellular domain that performs signaling functions (Cartellieri et al., Biomed Biotechnol 2010:956304, 2010). In many cases, the binding moiety is an antigen-binding fragment of a monoclonal antibody, such as a scFv, or a single domain antibody. Several different intracellular domains have been used to generate CARs. For example, the intracellular domain may consist of a signaling chain with an IT AM such as CD3ζ or FcRIγ. In some cases, the intracellular domain further includes at least one additional costimulatory domain such as the intracellular portion of CD28 and/or CD137.

BCMA antibodies and fragments thereof according to the present disclosure are engineered to include one or more binding moieties that specifically bind one or more targets of interest. Extracellular antigen-binding regions such as scFv or Fab can be part of the CAR that determines antigen specificity. The extracellular antigen binding region can bind to any complementary target, such as BCMA. In certain aspects of any of the embodiments disclosed herein, the extracellular antigen-binding region, such as a scFv, may comprise light chain CDRs specific for the antigen. The light chain CDRs may be the complementarity determining regions of the scFv light chain of an antigen binding unit such as a CAR. BCMA antibodies and fragments thereof encompass nucleic acids (eg, RNA and DNA), proteins (eg, antibodies), and combinations thereof.

CAR-expressing CTLs can be used to target specific cell types, such as tumor cells. Therefore, tumor antigen-specific monoclonal antibodies can be used to engineer CTLs expressing CARs containing antigen-binding fragments of the antigen-specific antibodies, thereby targeting the engineered CTLs to tumor cells expressing the tumor antigen. Engineered T cells have previously been used in adoptive therapy for some types of cancer (see, eg, Park et al., Mol Ther 15(4):825-833, 2007). The use of CAR-expressing T cells is more common than standard CTL-based immunotherapy because CAR-expressing CTL are not HLA-restricted and can therefore be used in any patient with a tumor expressing the target antigen.

Antibodies or fragments thereof as binding moieties

In some embodiments, the antibodies or fragments thereof described herein are anti-BCMA antibodies. In some cases, one or more of the binding moieties described herein is or includes an antibody, an antigen-binding fragment thereof, and/or an Fc region (or Fc fragment) thereof. The basic structure of an IgG antibody consists of two identical polypeptide light chains and two identical polypeptide heavy chains linked together by disulfide bonds. The first domain, located at the amino terminus of each chain, is variable in amino acid sequence and provides the antibody binding specificity found in each individual antibody. These are called variable heavy (VH) regions and variable light (VL) regions. The amino acid sequences of the other domains of each chain are relatively unchanged and are referred to as the constant heavy chain (CH) region and the constant light chain (CL) region. For IgG antibodies, the light chain consists of a A variable region (VL) and a constant region (CL). The IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2) and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).

Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic Antibodies, tetrameric antibodies containing two heavy chain molecules and two heavy chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chains - Antibody heavy chain pair, antibody endosome, antibody fusion (sometimes referred to herein as "antibody conjugate"), heterologous conjugated antibody, single domain antibody, monovalent antibody, single chain antibody or single chain Fv ( scFv), camelized antibody, affybody, Fab fragment, F(ab')2 fragment, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibody (including, for example, anti-anti-idiotypic ( anti-anti-Id), minibodies, domain antibodies, synthetic antibodies (sometimes referred to as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations.

As used herein, the term "Fc fragment" refers to one or more fragments of an Fc region that retains Fc function and/or activity as described herein, such as binding to an Fc receptor. As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al. (1989) Nature 341: 544-546), and isolated complementarity-determining regions (CDRs). Such antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for use in the same manner as intact antibodies.

In some aspects, the invention provides an antibody or fragment thereof that binds human BCMA, comprising a human heavy chain constant region and/or a light chain constant region, wherein the human heavy chain constant region comprises isotype variation A body comprising the Fc region of human IgGl, human IgG2, human IgG3 or human IgG4.

In another aspect, the invention provides a humanized antibody or fragment thereof that binds human BCMA, wherein the antibody comprises a variant human IgG Fc region comprising the amino acid substitution S324N, replacing the parent antibody with asparagine serine at amino acid position 324, and antibodies containing the variant human IgGFC region exhibited improved complement-dependent cytotoxicity (CDC) compared to the parent antibody.

Antibodies or fragments can be produced by any method known in the art for synthesizing antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using, for example, methods described in Morrison, 1985, Science 229:1202, and humanized antibodies can be produced, for example, by methods described in U.S. Patent No. 6,180,370.

Additional compositions and methods described herein are bispecific antibodies and multivalent antibodies, such as Segal et al., J. Immunol. Methods 248: 1-6 (2001); and Tutt et al., J. Immunol. 147: 60(1991).

Engineered antigen binding region

In some embodiments, the binding moiety is or includes an antibody (e.g., an IgG antibody, e.g., an IgG1, IgG2, or IgG3 antibody), or an antigen-binding fragment that is engineered to bind to one or more targets (i.e., antigens ) combine, that is, have different affinities. For example, an antibody can be engineered by modifying (eg, by addition, deletion, or substitution) amino acids within one or more of the antibody's CDRs and/or at positions related to the antibody's CDR structure. Exemplary non-limiting sites of antibodies that may be modified include the following (amino acid positions are indicated based on Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)).

In some embodiments, one or more of these disclosed amino acids can be modified by histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, Asparagine or glutamic acid substitution. Without wishing to be bound by theory, it is believed that substitution of amino acids at one or more of these positions with histidine can produce antibodies with pH-dependent antigen-binding properties. In some embodiments, non-histidine residues are substituted with histidine residues. In some embodiments, histidine residues are substituted with non-histidine residues. Additional engineered antigen binding regions include those described, for example, in U.S. Publication No. 20110229489.

engineered constant region

In some cases, the binding portion is or includes an antibody constant region, Fc region, or Fc fragment that binds one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptor).

In some cases, the binding moiety may be or include a constant region, Fc region, or Fc fragment of an IgG antibody engineered to include the amino acid residues described herein (e.g., 251-256, 285-290, 308 - Addition, deletion or substitution of one or more amino acids in 314, 385-389 and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)).

Generating and producing BCMA antibodies and fragments thereof

In some embodiments, BCMA antibody systems described herein are generated by immunizing humanized mice with human BCMA. In some embodiments, the antibodies or fragments thereof described herein are further engineered to include one or more binding moieties. For example, the sequence of a reference polypeptide (eg, a therapeutic antibody or a therapeutic fusion protein) can be obtained, and one or more amino acid residues can be added, deleted, or substituted. In some embodiments, one or more amino acid residues are modified by glycine, alanine, serine, cysteine, phenylalanine, tryptophan, tyrosine, proline, histidine , methionine, leucine, isoleucine, arginine, valine, lysine, aspartic acid, glutamic acid, threonine, asparagine or glutamine substitution . In a In some embodiments, substitution of the antibody comprising one or more amino acids enhances binding of the antibody to BCMA.

Antibodies can be produced recombinantly by first isolating the antibody and antibody-producing cells from a host animal, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another approach that can be taken is to express the antibody sequences in plants (eg, tobacco) or transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been disclosed. See, for example, Peeters et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock et al., J Immunol Methods 231:147, 1999. Methods for preparing antibody derivatives such as humanized, single chain, etc. are known in the art.

Measuring the interaction of the binding moiety with the target

The binding properties of the antibodies described herein or fragments thereof (e.g., anti-BCMA antibodies described herein) and targets (e.g., BCMA and/or FcRn) can be determined by methods known in the art, such as one of the following methods To measure: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis and scanning mutagenesis. Surface plasmon resonance (SPR) can be used to analyze binding interactions of antibodies with BCMA and/or FcRn. SPR or biomolecular interaction analysis (BIA) detects biologically specific interactions in real time without labeling any interactants. Changes in mass at the bonding surface of the BIA wafer (indicative of bonding events) cause changes in the refractive index of light near the surface. Changes in refractive index produce a detectable signal that is measured as an indication of real-time reactions between biomolecules. Methods using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705, and online resources provided by BIAcore International AB (Uppsala, Sweden). Additionally, KinExA® (Kinetic Exclusion Analysis) analysis available from Sapidyne Instruments (Boise, Id.) can also be used.

Information from SPR can be used to provide accurate estimates of the equilibrium dissociation constant (K _D ) and kinetic parameters (including K _on and K _off ) of binding moieties to targets (e.g., anti-BCMA antibodies to BCMA and/or FcRn). and quantitative measurement. Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of a specific binding moiety to the target at various pH levels can be evaluated. Variant amino acids at a given position can be identified that are associated with specific binding parameters (eg, high affinity, low affinity, and slow K _off ) at specific pH levels.

II.CAR Examples

Additionally, the present disclosure relates to methods and compositions in which BCMA CAR constructs are more suitable for use with NK cells because they have one or more components more relevant to NK cells than to other immune cells, including T cells. of biology.

In some embodiments, the invention relates to a chimeric antigen receptor (CAR) fusion protein comprising from the N-terminus to the C-terminus: (i) a single chain variable fragment (scFv) directed against BCMA (i.e., a "BCMA-binding (ii) a hinge region; (iii) a transmembrane domain; and (iv) one or more intracellular signaling domains, such as at least one costimulatory domain and an activation domain.

In particular embodiments, the present disclosure relates to reprogramming NK cells (eg, cord blood (CB)-derived NK cells) to target BCMA-expressing cancer cells. The present disclosure provides a number of novel CAR constructs that incorporate different BCMA scFv fused to the hinge region (especially the CD28 or IgG1 hinge region), the transmembrane domain, and the cytoplasmic domain including CD247 (also known as CD3ζ) and CD28 part of the signaling domain. In alternative embodiments, other costimulatory domains than CD28 are used.

BCMA conjugates

A suitable BCMA binder according to the invention may be a scFv that specifically binds BCMA. Typically, scFv can be in the form of VH-linker-VL or VL-linker-VH.

Specific linkers connecting the VH and VL chains can be used. An example of a linker amino acid sequence is as follows:

GGGGSGGGSGGGS ( SEQ ID NO: 15 ), or GGGGGGGGSGGSGGGGS ( SEQ ID NO: 16 ), or GGGGGGGGGGSGGGGGS ( SEQ ID NO: 17 ), or GGGGGGGSGGGGGS ( SEQ ID NO: 18 ), or GGTGGGGGTGTGTTCTGGGGGTGTGTGTGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGATCC ( SEQ ID NO: 24 ). In some embodiments, the linker comprises at least 75%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of SEQ ID NOs: 15-18 and 24 , an amino acid sequence that is at least 99% or 100% identical.

In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGSGGGSGGGGS ( SEQ ID NO: 16 ).

In some embodiments, the BCMA-binding region of the BCMA-CAR includes a variable heavy chain region (VH) and a variable light chain region (VL). In some embodiments, the BCMA conjugate contains three heavy chain complementarity determining regions (HCDR) in the VH and VL regions, namely HCDR1, HCDR2 and HCDR3, and three light chain complementarity determining regions (LCDR), namely LCDR1, LCDR2 and LCDR3.

In some embodiments, HCDR1 includes the amino acid sequence of SYAIH (SEQ ID NO:2), HCDR2 includes the amino acid sequence of VTWHDGSNKYYAESVMG (SEQ ID NO:3), and HCDR3 includes the amino acid sequence of AKFGEPQYFQH (SEQ ID NO:4) Amino acid sequence.

In some embodiments, LCDR1 includes the amino acid sequence of RASQGINNYLA (SEQ ID NO:6), LCDR2 includes the amino acid sequence of AASTLQS (SEQ ID NO:7), and LCDR3 includes the amino acid sequence of QQLKSYPFT (SEQ ID NO:8) Amino acid sequence.

In some embodiments, LCDR1 includes RASQGISSYLA (SEQ ID NO: 11) The amino acid sequence of LCDR2 includes the amino acid sequence of AASTLQS (SEQ ID NO:7), and LCDR3 includes the amino acid sequence of QQLNSYPFT (SEQ ID NO:12).

In some embodiments, LCDR1 comprises the amino acid sequence of RASQGISSYLA (SEQ ID NO: 11), LCDR2 comprises the amino acid sequence of AASTLQS (SEQ ID NO: 7), and LCDR3 comprises the amino acid sequence of QQLNSYPWT (SEQ ID NO: 14) Amino acid sequence.

In some embodiments, the heavy chain variable region (VH) of the BCMA conjugate comprises the following amino acid sequence:

(SEQ ID NO：1)。

( SEQ ID NO: 1 ).

It is contemplated that any amino acid substitution at any position other than the CDR sequence may be changed to another amino acid, such as a conservative amino acid substitution (as defined herein). In some embodiments, the VH comprises a sequence 70% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 75% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 80% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 85% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 90% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 95% identical to SEQ ID NO: 1. In some embodiments, the VH comprises a sequence 99% identical to SEQ ID NO: 1.

In some embodiments, the heavy chain variable region (VH) of the BCMA conjugate comprises the following amino acid sequence:

(SEQ ID NO：9)。

( SEQ ID NO:9 ).

It is contemplated that any amino acid substitution at any position other than the CDR sequence may be changed to another amino acid, such as a conservative amino acid substitution (as defined herein). In some embodiments, the VH comprises a sequence 70% identical to SEQ ID NO:9. In some embodiments, the VH comprises a sequence 75% identical to SEQ ID NO:9. In some embodiments, the VH comprises a sequence 80% identical to SEQ ID NO: 9. In some embodiments, the VH comprises a sequence 85% identical to SEQ ID NO: 9. In some embodiments, the VH comprises a sequence 90% identical to SEQ ID NO: 9. In some embodiments, the VH comprises a sequence 95% identical to SEQ ID NO:9. In some embodiments, the VH comprises a sequence 99% identical to SEQ ID NO: 9.

In some embodiments, the BCMA conjugate comprises a light chain variable region (VL). In some embodiments, VL comprises the following amino acid sequence:

DIVMTQSPSFLSASVGDRVTITCRASQGINNYLAWYQQKPGIAPKLLIYAASTLQSGVPSRFGGSGSGTEFTLTISSLQPEDFATYYCQQLKSYPFTFGPGTKVEIK ( SEQ ID NO: 5 ).

It is contemplated that any amino acid substitution at any position other than the CDR sequence may be changed to another amino acid, such as a conservative amino acid substitution (as defined herein). In some embodiments, VL comprises a sequence 70% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 75% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 80% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 85% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 90% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 95% identical to SEQ ID NO: 5. In some embodiments, VL comprises a sequence 99% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-binding region of the CAR includes a light chain variable region (VL). In some embodiments, VL comprises the following amino acid sequence:

DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPFTFGPGTKVDIK ( SEQ ID NO: 10 ).

It is contemplated that any amino acid substitution at any position other than the CDR sequence may be changed to another amino acid, such as a conservative amino acid substitution (as defined herein). In some embodiments, VL comprises a sequence 70% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 75% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 80% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 85% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 90% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 95% identical to SEQ ID NO: 10. In some embodiments, VL comprises a sequence 99% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-binding region of the CAR includes a light chain variable region (VL). In some embodiments, VL comprises the following amino acid sequence:

(SEQ ID NO：13)。

( SEQ ID NO: 13 ).

It is contemplated that any amino acid substitution at any position other than the CDR sequence may be changed to another amino acid, such as a conservative amino acid substitution (as defined herein). In some embodiments, VL comprises a sequence 70% identical to SEQ ID NO: 13. In some embodiments, VL comprises a sequence 75% identical to SEQ ID NO: 13. In some embodiments, VL comprises a sequence 80% identical to SEQ ID NO: 13 List. In some embodiments, VL comprises a sequence 85% identical to SEQ ID NO: 13. In some embodiments, VL comprises a sequence 90% identical to SEQ ID NO: 13. In some embodiments, VL comprises a sequence 95% identical to SEQ ID NO: 13. In some embodiments, VL comprises a sequence 99% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 70% identical to SEQ ID NO: 1 and a VL that is at least 70% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 75% identical to SEQ ID NO: 1 and a VL that is at least 75% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 80% identical to SEQ ID NO: 1 and a VL that is at least 80% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 90% identical to SEQ ID NO: 1 and a VL that is at least 90% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 95% identical to SEQ ID NO: 1 and a VL that is at least 95% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 97% identical to SEQ ID NO: 1 and a VL that is at least 97% identical to SEQ ID NO: 5.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 70% identical to SEQ ID NO: 9 and a VL that is at least 70% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 75% identical to SEQ ID NO: 9 and a VL that is at least 75% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 80% identical to SEQ ID NO: 9 and a VL that is at least 80% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 90% identical to SEQ ID NO: 9 and a VL that is at least 90% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 95% identical to SEQ ID NO: 9 and a VL that is at least 95% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 97% identical to SEQ ID NO: 9 and a VL that is at least 97% identical to SEQ ID NO: 10.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 70% identical to SEQ ID NO: 1 and a VL that is at least 70% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 75% identical to SEQ ID NO: 1 and a VL that is at least 75% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 80% identical to SEQ ID NO: 1 and a VL that is at least 80% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 90% identical to SEQ ID NO: 1 and a VL that is at least 90% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 95% identical to SEQ ID NO: 1 and a VL that is at least 95% identical to SEQ ID NO: 13.

In some embodiments, the BCMA-conjugate in the chimeric antigen receptor comprises a VH that is at least 97% identical to SEQ ID NO: 1 and a VL that is at least 97% identical to SEQ ID NO: 13.

In some embodiments, an embodiment, a specific embodiment, the molecule that specifically binds the antigen binds with a dissociation constant ( _Kd ) of about 1×10 ⁻⁷ M. In some embodiments, the antigen-binding molecule specifically binds the antigen with "high affinity" with a _Kd of about 1×10 ^-9 M to about 5×10 ^-9 M. In some embodiments, the antigen-binding molecule specifically binds the antigen with "very high affinity" with a _Kd of 1×10 ^-10 M to about 5×10 ^-10 M. In one embodiment, the antigen-binding molecule has a _Kd of 10 ^-9 M. In one embodiment, the dissociation rate is less than about 1×10 ⁻⁵ . In other embodiments, the antigen-binding molecule binds human BCMA with a _K between about 1×10 ⁻⁷ M and about 1×10 ⁻¹³ M. In another embodiment, the antigen-binding molecule binds human BCMA with a _K of about 1×10 ⁻¹⁰ M to about 5×10 ⁻¹⁰ M.

In another specific embodiment, the molecule that specifically binds the antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, the molecule that specifically binds the antigen does not cross-react with other non-BCMA proteins. In a specific embodiment, provided herein are antibodies, or fragments thereof, that bind BCMA with greater affinity than another unrelated antigen. In certain embodiments, provided herein are antibodies or fragments thereof that bind BCMA (e.g., human BCMA) with an affinity that is 20%, 25%, 30%, 35%, 40%, 45% higher than binding another unrelated antigen. %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher, as determined by, for example, radioimmunoassay, surface plasmon resonance or kinetic Learn to exclude analytical measurement. In a specific embodiment, an anti-BCMA antibody or antigen-binding fragment thereof described herein binds to an unrelated non-BCMA protein to a degree that is less than 10%, 15%, or 20% of the antibody's binding to the BCMA protein, as determined by, e.g., radioimmunoassay. Analytical measurement.

In a specific embodiment, provided herein are antibodies or fragments thereof that bind human BCMA with higher affinity than another species of BCMA. In certain embodiments, provided herein are antibodies, or fragments thereof, that bind human BCMA with greater affinity than another species of BCMA 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher, as by e.g. radiation Immunoassay, surface plasmon resonance or kinetic exclusion analysis measurements. In a particular embodiment, an antibody or fragment thereof described herein that binds human BCMA will bind to human BCMA at a lower concentration than that of an antibody or fragment thereof, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion analysis. 10%, 15% or 20% of the human BCMA protein binds to another BCMA protein.

hinge area

In certain embodiments, the CAR polypeptide includes an extracellular spacer domain (also referred to as a hinge) connecting the antigen-binding domain and the transmembrane domain. The hinge domain provides a spacer between the scFv and the cell membrane and intracellular signaling modules that mediate NK cell activation or T cell activation. The extracellular spacer domain may include, but is not limited to, hinges from human proteins. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge, or a CD8a hinge; an artificial spacer made of a polypeptide, such as Gly3; or an IgG (eg, human IgGl or IgG4) CH1, CH2 and/or CH3 domains.

In certain cases, the extracellular spacer domain may comprise (i) the hinge region, CH2 and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge region and CH2 of IgG4, (iv) the hinge of CD8-α Region, (v) hinge region of CD28, (vi) hinge region, CH2 and CH3 region of IgG1, (vii) hinge region of IgG1.

A particularly useful hinge according to the present invention is derived from CD28. In certain embodiments, a CAR polypeptide comprises a specific CD28 hinge amino acid sequence or is encoded by a specific CD28 hinge nucleic acid sequence. Examples are as follows:

Exemplary suitable CD28 hinges contain the following amino acid sequences:

RAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKDPK ( SEQ ID NO: 36 )

Exemplary suitable CD28 hinges are encoded by the following nucleic acid sequences:

cgggcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccaaagatcccaaa ( SEQ ID NO: 35 )

Exemplary suitable IgG hinges contain the following amino acid sequences:

(SEQ ID NO：37)

( SEQ ID NO: 37 )

Exemplary suitable IgG hinges contain the following nucleic acid sequences:

(SEQ ID NO：38)

( SEQ ID NO: 38 )

transmembrane domain

Transmembranely connects the intracellular signaling domain to the hinge region of the CAR. In some embodiments, the CAR includes a transmembrane domain. In some embodiments, suitable transmembrane domains according to the invention are the following Transmembrane domains: CD28, 4-1BB/CD137, CD8 (e.g., CD8 alpha), CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 , CTLA4, PD-1 or CD154. Exemplary transmembrane domains are disclosed in WO2020227446, which is incorporated herein in its entirety.

In some embodiments, the CD28 transmembrane domain is associated with the transmembrane domain. In some embodiments, the transmembrane domain is a CD28 transmembrane domain comprising the following amino acid sequence:

FWVLVVVGGVLACYSLLVTVAFIIFWV( SEQ ID NO: 26 )

In some embodiments, the transmembrane is encoded by the following nucleic acid sequence:

ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg ( SEQ ID NO: 27 )

costimulatory domain

A CAR may contain one or more costimulatory domains. Costimulatory signals are required to achieve robust chimeric antigen receptors (CARs), including cell expansion, function, persistence, and anti-tumor activity. In some embodiments, costimulatory regions according to the invention are the following signaling regions: CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD- 1), Inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), CD3 γ, CD3 δ, CD3 ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, interleukin receptor, integrin, signaling Conducting lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, CD11b, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9( CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT , GADS, SLP-76, PAG/Cbp, CD19a, ligands that specifically bind CD83, or any combination thereof.

In some embodiments, the costimulatory domain includes:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS ( SEQ ID NO: 28 )

In some embodiments, the costimulation domain is encoded by:

aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctca ( SEQ ID NO: 29 )

activation domain

According to the present invention, the CAR construct may also include an activation domain. In specific embodiments, a CAR polypeptide comprises an immune cell activation moiety. The activation moiety and costimulatory moiety work together to activate downstream signaling cascades, leading to NK cell activation, proliferation, acquisition of effector functions, and secretion of inflammatory cytokines and chemokines. In some embodiments, the activating moiety is CD3ζ.

An example of the CD3ζ amino acid sequence is as follows:

(SEQ ID NO：30)

( SEQ ID NO: 30 )

An example of a CD3ζ nucleic acid sequence is as follows:

(SEQ ID NO：31)

( SEQ ID NO: 31 )

signal peptide

In specific embodiments, the CAR polypeptide comprises a signal peptide. The signal peptide is part of the extracellular domain of the CAR polypeptide. In some embodiments, the extracellular domain is the portion of the CAR protein that is located outside the cytoplasm and exposed to the extracellular space. The function of signal peptide is to transfer the recognized protein signal to the endoplasmic reticulum of cells. In some embodiments, the signal peptide can be selected from the group consisting of heavy chain signal peptide, IL-15 signal peptide, CD8a signal peptide, and GMCSF-R signal peptide.

An example of a signal peptide amino acid sequence is as follows:

MEFGLSWLFLVAILKGVQC( SEQ ID NO:51 )

An example of a signal peptide nucleic acid sequence is as follows:

atggaattcggattgtcatggttgttcctcgtcgcaattctcaagggcgtgcagtgc ( SEQ ID NO: 52 ).

An example of a signal peptide amino acid sequence is as follows:

MRISKPHLRSISIQCYLCLLLNSHFLTEA ( SEQ ID NO: 32 )

An example of a signal peptide nucleic acid sequence is as follows:

Atgcgcattagcaagccccacctgcggagcatcagcatccagtgctacctgtgcctgctgctgaacagccacttcctgaccgaggcc ( SEQ ID NO: 33 ).

In some embodiments, the CAR contains a cleavage site. An example of a cleavage site is:

GPQCTNYALLKLAGDVESNPGP ( SEQ ID NO: 39 ).

In some embodiments, the cleavage site is encoded by:

ggaccgcagtgtactaattatgctctcttgaaattggctggagatgttgagagcaatcccgggccc ( SEQ ID NO: 40 ).

interleukin

In some embodiments, expression of interleukins in CAR-expressing cells enhances their anti-tumor efficacy. In one embodiment, the interleukin is expressed as part of the CAR. In another embodiment, the interleukin is expressed in a separate expression system. In some embodiments, the interleukin may be selected from IL-15, IL-12, IL-2, IL-18, IL-21, or a combination thereof. In one embodiment, the interleukin is selected from the group consisting of growth hormones, such as human growth hormone, N-methionine human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; Prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placenta Prolactin; Mullerian-inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF- β; platelet growth factors; transforming growth factors (TGFs), such as TGF-α and TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon -α, -β, and -γ; colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G- CSF); interleukins (IL), such as IL-1, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-15; tumor necrosis factors, such as TNFα or TNF-β; and other peptide factors, including LIF and kit ligand (KL).

In one embodiment, the interleukin is IL-15. In one embodiment, the interleukin IL-15 region includes the following amino acid sequence:

(SEQ ID NO：23)。

( SEQ ID NO: 23 ).

An example of an IL-15 nucleic acid sequence is as follows:

(SEQ ID NO：34)

( SEQ ID NO: 34 )

In some embodiments, IL-15 polypeptides contemplated by the present disclosure may comprise SEQ ID NO: 23 or be at least 70%, 75%, 80%, 85%, 90%, 95%, 96% identical to SEQ ID NO: 23 , 97%, 98%, 99% or more % identical sequences.

suicide gene

In certain embodiments, suicide genes are used in conjunction with any kind of cell therapy to control its use and allow termination of the cell therapy at a desired event and/or time. Suicide genes are used in transduced cells with the aim of triggering the death of the transduced cells when needed. Antigen-targeted cells of the disclosure that have been modified to contain vectors contemplated by the disclosure may include one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that upon administration of a prodrug or other agent achieves conversion of its gene product into a compound that kills its host cell. In other embodiments, the suicide gene encodes a gene product that, when desired If desired, the gene product is targeted by an agent (such as an antibody) that targets the suicide gene product. "Suicide gene product" describes the protein or polypeptide encoded by the suicide gene.

Examples of suicide gene/prodrug combinations that can be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytarabine. Escherichia coli ( E.coli ) purine nucleoside phosphorylase, a so-called suicide gene, can be used to convert the prodrug 6-methylpurine deoxynucleoside into the toxic purine 6-methylpurine. Other examples of suicide genes used with prodrug therapy are the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

Exemplary suicide genes also include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated form of EGFR variant III (EGFRv3) can be used as a suicide antigen ablated by cetuximab. Other suicide genes known in the art that may be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxyl esterase (CE), nitrate base reductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine-α,γ-lyase (MET) and thymidine phosphorylase (TP).

In some embodiments, inducible caspase 9 (iC9) is used as an exemplary suicide gene. One example iC9 is described, for example, in Yagyu S et al. Mol Ther. 2015 Sep;23(9):1475-85, which is incorporated herein by reference in its entirety. In some embodiments, iCaspase9 includes the following amino acid sequence:

(SEQ ID NO：25)。

( SEQ ID NO: 25 ).

In some embodiments, the present disclosure relates to providing methods and compositions for terminating cell therapy using non-cleavable mutants of 26kd TNFα. TNFα mutant systems are cleavable, making them membrane-bound and non-secretable. Cells expressing non-cleavable TNFα mutants can be targeted for selective deletion, including, for example, using currently clinically approved FDA-approved TNFα antibodies, such as etanercept, infliximab, or adalimumab. Mutated TNFα polypeptides may be co-expressed with one or more therapeutic transgenes, such as a gene encoding a CAR. Furthermore, cells expressing TNF-α mutants have superior activity against tumor targets, which is mediated by the biological activity of the membrane-bound TNFα protein.

In a specific embodiment, the suicide gene is a tumor necrosis factor (TNF) alpha mutant that cannot be cleaved by standard enzymes that naturally cleave TNF, such as TNF alpha converting enzyme (also known as TACE). Thus, in certain embodiments, TNFα mutants are membrane-bound and non-secretable. TNFα mutants used in the present disclosure can be targeted by one or more agents (including at least one antibody) that bind to the mutant, such that upon binding of the agent to the TNFα mutant on the cell surface, the cell dies. Embodiments of the present disclosure allow for the use of TNF[alpha] mutants as markers for cells expressing it.

Cells expressing non-cleavable TNFα mutants can be targeted for selective deletion, including, for example, using currently clinically approved FDA-approved TNFα antibodies, such as etanercept, infliximab, or adalimumab. Mutated TNFα polypeptides may be co-expressed with one or more therapeutic transgenes in the cell, such as genes encoding CARs, including BCMA-targeting CARs. Furthermore, cells expressing TNF-α mutants have superior activity against tumor targets, which is mediated by the biological activity of the membrane-bound TNFα protein.

Wild-type TNFα has a 26kD transmembrane form and a 17kD secretory component. In some embodiments, TNFα mutants described in Perez et al. (1990) can be used in the present disclosure. In specific embodiments, the TNFα mutant is comprised at positions -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or combinations thereof The corresponding amino acid is missing. Specific combinations include deficiencies in Loss: -3 up to and including 13; -3 up to and including 12; -3 up to and including 11; -3 up to and including 10; -3 up to and including 9; -3 up to and including 8; -3 up to and including 7 ; -3 to and including 6; -3 to and including 5; -3 to and including 4; -3 to and including 3; -3 to and including 2; -3 to and including 1; -3 to and including -1 ; -3 to and inclusive -2; -2 to and inclusive 13; -2 to and inclusive 12; -2 to and inclusive 11; -2 to and inclusive 10; -2 to and inclusive 9; -2 to and inclusive 8 ; -2 to and including 7; -2 to and including 6; -2 to and including 5; -2 to and including 4; -2 to and including 3; -2 to and including 2; -2 to and including 1; -2 to and including -1; -1 to and including 13; -1 to and including 12; -1 to and including 11; -1 to and including 10; -1 to and including 9; -1 to and including 8; -1 to and including 7; -1 to and including 6; -1 to and including 5; -1 to and including 4; -1 to and including 3; -1 to and including 2; -1 to and including 1; 1 to and including 13; 1 to and including 12; 1 to and including 11; 1 to and including 10; 1 to and including 9; 1 to and including 8; 1 to and including 7; 1 to and including 6; 1 to and including including 5; 1 to and including 4; 1 to and including 3; 1 to and including 2; and so on. In specific embodiments, examples of TNF-α mutants of the present disclosure include at least the following deletions for 17kD TNF: (1) Val1 deletion and Prol12 deletion; (2) Val13 deletion; (3) Val1 deletion and Val13 deletion; (4) Val1 up to and including Pro12 deletion and Val13 deletion (deletion 13aa); (5) Ala-3 up to and including Val 13 deletion (deletion 16 aa).

TNFα mutants can be produced by any suitable method, but in certain embodiments, they are produced by site-directed mutagenesis. In some cases, TNFα mutants may have mutations in addition to those that render the protein non-cleavable. In certain cases, a TNFα mutant may have 1, 2, 3 or more mutations in addition to deletions at Val, Pro12, and/or Val13, or regions in between. Mutations other than those rendering the mutant non-secretable may be one or more of amino acid substitutions, deletions, additions, inversions, and the like. For example, where the additional mutation is an amino acid substitution, the substitution may or may not be a conservative amino acid. In some cases, the N-terminus and/or C-terminus of the protein may exist 1, 2, 3, 4, 5 or more additional amino acids. In some cases, a TNFα mutant has (1) one or more mutations that render the mutant non-secretable; (2) one or more mutations that prevent the mutant's outside-in signaling; and/or (3) one or Multiple mutations that interfere with the binding of the mutant to TNF receptor 1 and/or TNF receptor 2.

In specific embodiments, the TNFα mutant polypeptide includes the following deletions with respect to SEQ ID NO: 25: amino acid residue 1 and amino acid residue 12; amino acid residue 1 and amino acid residue 13; amino group Acid residues 1-12; amino acid residues 1-13; or amino acid residues -1 to 13.

Exemplary TNFα mutant polypeptide sequences, mutants and variants are disclosed in WO2020106619 and WO2021055349, which are incorporated herein by reference in their entirety.

Exemplary full-length BCMA-CAR sequence

In some embodiments, BCMA-CAR comprises an amino acid sequence comprising:

(SEQ ID NO：19)。

( SEQ ID NO: 19 ).

In some embodiments, the BCMA-CAR comprises at least 70% of the same as SEQ ID NO: 19 Identical amino acid sequence. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 19. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 19. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 19. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. In some embodiments, a BCMA-CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19. In some embodiments, a BCMA-CAR comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 19. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 19.

In some embodiments, BCMA-CAR comprises a nucleic acid sequence comprising:

(SEQ ID NO：42)。

( SEQ ID NO: 42 ).

In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 42. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 42. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 42.

In some embodiments, BCMA-CAR is connected to IL-15 via a linker and a cleavage peptide, and the IL-15 includes SEQ ID NO: 45:

(SEQ ID NO：45)

( SEQ ID NO: 45 )

BCMA-CAR linked to IL-15 represented by SEQ ID NO: 45 is sometimes referred to as BCMA28-1 in this specification. Another BCMA-CAR linked to IL-15, in which the CD28 hinge is replaced by an IgG1 hinge, is sometimes referred to as BCMAIg1 in this specification.

In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 45. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 45.

In some embodiments, BCMA-CAR-IL-15 includes:

(SEQ ID NO：46)。

( SEQ ID NO: 46 ).

In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 46. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 46.

In some embodiments, BCMA-CAR comprises an amino acid sequence comprising:

(SEQ ID NO：20)

( SEQ ID NO:20 )

In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, a BCMA-CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 20. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 20.

In some embodiments, BCMA-CAR comprises a nucleic acid sequence comprising:

(SEQ ID NO：43)

( SEQ ID NO: 43 )

In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 43. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 43. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 43. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 43. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 43. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 43. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 43. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 43.

In some embodiments, BCMA-CAR is linked to IL-15 via a linker and a cleavage peptide, and the IL-15 includes SEQ ID NO: 47:

(SEQ ID NO：47)

( SEQ ID NO: 47 )

BCMA-CAR linked to IL-15 represented by SEQ ID NO: 47 is sometimes referred to as BCMA28-2 in this specification. Another BCMA-CAR linked to IL-15, in which the CD28 hinge is replaced by an IgG1 hinge, is sometimes referred to as BCMAIg2 in this specification.

In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 47. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 47.

In some embodiments, BCMA-CAR-IL-15 includes:

(SEQ ID NO：48)

( SEQ ID NO: 48 )

In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 48. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 48.

In some embodiments, BCMA-CAR comprises an amino acid sequence comprising:

(SEQ ID NO：21)

( SEQ ID NO: 21 )

In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 21. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 21. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 21. In some embodiments, BCMA-CAR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the BCMA-CAR comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 21. In some embodiments, a BCMA-CAR comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 21.

In some embodiments, BCMA-CAR comprises a nucleic acid sequence comprising:

(SEQ ID NO：44)

( SEQ ID NO: 44 )

In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 44. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 44. In some embodiments, BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 44. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 44. In some embodiments, BCMA-CAR comprises at least 90% the same as SEQ ID NO: 44 Identical nucleic acid sequence. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 44. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 44. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 44.

In some embodiments, BCMA-CAR is connected to IL-15 via a linker and a cleavage peptide, and the IL-15 includes SEQ ID NO: 49:

(SEQ ID NO： 49)

( SEQ ID NO: 49 )

In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 49. In some embodiments, BCMA-CAR-IL15 comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 49.

In some embodiments, BCMA-CAR-IL-15 includes:

(SEQ ID NO：50)

( SEQ ID NO:50 )

In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises SEQ ID NO: 50 Nucleic acid sequences that are at least 75% identical. In some embodiments, a BCMA-CAR comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 50. In some embodiments, BCMA-CAR-IL15 comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 50.

Embodiments of the disclosure encompass cells expressing one or more CARs and one or more suicide genes as contemplated herein. In certain embodiments, NK cells comprise recombinant nucleic acids encoding one or more CARs and one or more engineered non-secreted, membrane-bound TNF-alpha mutant polypeptides. In certain embodiments, in addition to expressing one or more CAR and TNF-alpha mutant polypeptides, the cells also comprise nucleic acids encoding one or more therapeutic gene products.

carrier

In certain aspects, provided herein are vectors comprising polynucleotides of the invention. In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an AAV vector, or a retroviral vector. In some embodiments, the vector can be a viral vector. Examples of viral vectors include at least retroviral, lentiviral, adenoviral or adeno-associated viral vectors. Examples of non-viral vectors include at least plastids, transposons, lipids, nanoparticles, and the like.

When an immune cell line is transduced with a vector encoding a genetically engineered receptor, and it is also necessary to transduce one or more genes into the cells, such as suicide genes and/or interleukins and/or optional therapeutic gene products. Under this condition, the antigen-targeting receptor, suicide gene, interleukin and optional therapeutic gene may or may not be included on or in the same vector. In some cases, CAR, suicide genes The factors, interleukins and optional therapeutic genes are expressed by the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the CAR, suicide gene, interleukin, and optional therapeutic gene may or may not be regulated by the same regulatory elements. When the CAR, suicide gene, interleukin, and optional therapeutic gene are located on the same vector, they may or may not appear as separate polypeptides. For example, where they are expressed as separate polypeptides, they may be separated on the vector by a 2A element or an IRES element (or both types may be used once or more than once on the same vector).

In some embodiments, the interleukin and suicide gene are represented by the same polypeptide, wherein they are separated by a 2A element. In some embodiments, the 2A element can induce ribosome skipping during protein translation in the cell.

In some embodiments, the 2A element includes the following amino acid sequence:

QCTNYALLKLAGDVESNPGP ( SEQ ID NO: 53 ).

cells

The present disclosure encompasses any type of immune cell or stem cell that has at least one vector encoding a genetically engineered receptor that includes a BCMA CAR that includes a CD28 hinge domain. In some cases, different vectors encode the CAR and encode the suicide gene and/or interleukin. Immune cells, including NK cells, can be derived from umbilical cord blood (including pooled cord blood from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. For example, NK cells can be derived from cell lines such as, but not limited to, NK-92 cells. The NK cells can be cord blood mononuclear cells, such as CD56 ⁺ NK cells.

This disclosure covers any type of immune cells or other cells, including conventional T cells, gamma-delta T cells, NKT and invariant NK T cells, regulatory T cells, macrophages, B cells, dendrites cells, mesenchymal stromal cells (MSC), or mixtures thereof.

NK cells are a key component of the innate immune response and an important player in the first line of defense against malignant cells. The ability of NK cells to kill malignant cells without prior sensitization helps them to work quickly, unlike T cells, which need to recognize tumor antigens presented in the context of HLA molecules.

In some cases, the NK cells have been expanded in the presence of an effective amount of universal antigen presenting cells (UAPC), including in any suitable ratio. Cells can be cultured with UAPC, for example, in a ratio of 10:1 to 1:10; 9:1 to 1:9; 8:1 to 1:8; 7:1 to 1:7; 6:1 to 1:6; 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 2:1 to 1:2; or 1:1, including a ratio of 1:2. In some cases, NK cells expand in the presence of IL-2, such as at concentrations of 10-500U/mL, 10-400U/mL, 10-300U/mL, 10-200U/mL, 10-100U/mL, 10 -50U/mL, 100-500U/mL, 100-400U/mL, 100-300U/mL, 100-200U/mL, 200-500U/mL, 200-400U/mL, 200-300U/mL, 300-500U /mL, 300-400U/mL or 400-500U/mL.

After genetic modification with a vector, NK cells can be immediately infused or stored. In some aspects, after genetic modification, cells can reproduce in vitro as a large population for days, weeks, or weeks, about 1, 2, 3, 4, 5, or more days after the gene is transferred to the cell. moon. In another aspect, transfectants are selected and expression cassettes or plasmids are selected to demonstrate the presence of a single integration or episomal maintenance, and expression of the CAR is amplified ex vivo.

Embodiments of the disclosure encompass cells expressing one or more CARs and one or more suicide genes as contemplated herein. In certain embodiments, NK cells comprise recombinant nucleic acids encoding one or more CARs and one or more engineered non-secreted, membrane-bound TNF-alpha mutant polypeptides. In certain embodiments, in addition to expressing one or more CAR and TNF-alpha mutant polypeptides, the cells also comprise nucleic acids encoding one or more therapeutic gene products.

Cells may be obtained directly from an individual or may be obtained from a depository or other storage facility. The cells used as therapy may be autologous or allogeneic to the individual to whom the cells are provided as therapy.

Cells can be derived from an individual in need of treatment of a medical condition and manipulated to express a CAR, optionally a suicide gene, optionally an interleukin, and optionally a therapeutic gene product (e.g., using standard transduction techniques and the expansion of adoptive cell therapy), they may be provided to the individual from whom they were originally derived. In some cases, cells are stored for later use by the individual or another individual.

Immune cells may be included in a population of cells, and the population may have a majority of cells transduced with one or more receptors and/or one or more suicide genes and/or one or more interleukins. The cell population may comprise 51%, 52%, 53%, 54%, 55%, 56%, 57 transduced with one or more CARs and/or one or more suicide genes and/or one or more interleukins. %, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of immune cells. One or more CARs and/or one or more suicide genes and/or one or more interleukins may be separate polypeptides.

Immune cells may be generated with one or more CARs and/or one or more suicide genes and/or one or more interleukins for modularization for specific purposes. For example, cells can be generated, including for commercial distribution, expressing the CAR and/or one or more suicide genes and/or one or more interleukins (or distributed together with nucleic acid encoding the mutant for subsequent transduction) , and users can modify it to express one or more other genes of interest (including therapeutic genes) depending on their intended purpose. For example, an individual interested in treating antigen-positive cells (including antigen-positive cancers or cells infected with infectious agents) can obtain or generate suicide gene-expressing cells (or allogeneic interleukin-expressing cells) and modify them to express antibodies containing receptor for the original specific scFv, or vice versa.

In certain embodiments, NK cells are used, and the genome of the transduced NK cell can be modified to express one or more CARs and/or one or more suicide genes and/or one or more interleukins. The genome may be modified in any manner, but in certain embodiments, the genome is modified, for example, by CRISPR gene editing. The genome of a cell can be modified to enhance the cell's effectiveness for any purpose.

As a non-limiting example, NK cells may express a BCMA-binding CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21, 45, 47, and 49. In some examples, an NK cell can comprise a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42-44, 46, 48, and 50.

III. Treatment methods

In various embodiments, diseased cells expressing a desired target on their surface are targeted in order to ameliorate a medical condition in an individual suffering from a medical condition or to reduce the risk or delay the severity and/or onset of a medical condition in an individual. or other cells. In certain circumstances, in order to kill cancer cells, cancer cells expressing endogenous antigens are targeted. In other cases, cells infected by infectious agents are targeted in order to kill infected cells.

In some embodiments, an antibody or fragment thereof described herein (eg, an anti-BCMA antibody as described herein) is used in a method of treating one or more BCMA-related disorders. In some embodiments, an antibody or fragment thereof described herein (eg, an anti-BCMA antibody as described herein) is used as a drug. BCMA-related disorders may include, but are not limited to, disorders caused by, including, symptoms caused in whole or in part by, or known to occur with, BCMA manifestations.

According to the present disclosure, the antibodies, fragments thereof, and CARs and compositions described herein can be used to treat BCMA-related cancers. Cancer is a broad group of diseases characterized by abnormalities in the body Uncontrolled growth of cells. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. In some embodiments, "cancer" or "cancerous tissue" includes solid tumors. Examples of cancers that may be treated by the methods of the invention include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myeloma and other leukocyte malignancies.

In some aspects, the invention provides methods for treating cancer comprising administering an agent that binds BCMA (eg, an anti-BCMA antibody or fragment thereof described herein).

In various embodiments, administration of an antibody or fragment thereof described herein (e.g., an anti-BCMA antibody or fragment thereof described herein) results in BCMA-related disorders as described herein or otherwise known in the art. A reduction in the incidence, frequency, level and/or amount of one or more symptoms or biomarkers, such as a reduction of at least 3%, 4, or 4 in one or more symptoms or biomarkers compared to previously measured values in an individual or compared to a reference value %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

In some embodiments, administration of an antibody described herein or a fragment thereof (e.g., an anti-BCMA antibody described herein) to an individual with cancer results in one or more symptoms or biomarkers of cancer that are greater than a reference antibody (e.g., Antibodies that cross compete for BCMA binding) greater reduction or improvement under comparable conditions.

In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-BCMA antibody described herein) can be administered at an increased dosage amount compared to a reference protein (e.g., an antibody that cross-competes for BCMA binding) while Achieving equivalent, equally effective, comparably effective or substantially effective results wherein the anti-BCMA antibody is formulated in the same, equivalent or substantially equivalent form as the reference (e.g., an antibody that cross-competes for BCMA binding) and/or by Administer via the same, equivalent or substantially equivalent route of administration. In some embodiments, anti-BCMA antibodies described herein can cross-compete BCMA with a reference antibody (e.g., combined antibodies) administered at increased intervals while achieving equal, equally effective, comparably effective or substantially effective results, wherein the anti-BCMA antibody is in the same, equivalent or substantially equivalent formulation as the reference and /or administered through the same, equivalent or substantially equivalent administration route. In some embodiments, the anti-BCMA antibodies described herein can be administered with a reduced number of unit doses and/or a reduced treatment period compared to a reference antibody while achieving equal, equally effective, comparably effective, or substantially effective As a result, wherein the anti-BCMA antibody is administered in the same, equivalent, or substantially equivalent formulation and/or by the same, equivalent, or substantially equivalent route of administration as the reference (e.g., an antibody that cross-competes for BCMA binding) Make the investment.

According to some such embodiments, an administered dose of an anti-BCMA antibody described herein is less likely to elicit an adverse reaction when administered to a subject than an effective dose of a reference antibody (e.g., an antibody that cross-competes for BCMA binding) , such as adverse immune reactions. Thus, in various embodiments, an anti-BCMA antibody as disclosed herein is less likely than a reference antibody to induce adverse reactions or side effects per unit of activity administered. In various embodiments, an anti-BCMA antibody as disclosed herein is less likely than a reference antibody to induce adverse reactions or side effects of a particular severity per unit of activity administered. In various embodiments, an anti-BCMA antibody as disclosed herein induces one or more adverse reactions or side effects to a lower extent or in fewer patients than a reference antibody per unit of activity administered. Examples of adverse reactions or side effects that may be associated with administration of antibodies capable of binding BCMA may include headache, nasopharyngitis, back pain, nausea, diarrhea, hypertension, upper respiratory tract infection, abdominal pain, vomiting, anemia, cough, peripheral edema, and /or urinary tract infection.

In some embodiments, upon administration to an individual (e.g., in a single dose), an antibody described herein or a fragment thereof (e.g., an anti-BCMA antibody described herein) is administered at a defined time after administration (e.g., 1, Water in plasma measured at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days) The level increases relative to a control (e.g., an antibody that cross-competes for BCMA binding) at the same defined time. For example, at a defined time after administration of a single dose, the level of an anti-BCMA antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70% higher than the corresponding level of the reference antibody , 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500%.

In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-BCMA antibody described herein) is administered (e.g., in a single dose) at a defined time (e.g., 1, 2, 3, 4, Levels in plasma measured at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days) are increased relative to levels in controls at the same defined time. For example, at a defined time after administration, the level of an anti-BCMA antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% higher than the corresponding level of the reference antibody. , 90%, 100%, 150%, 200%, 300%, 400% or 500%.

In some embodiments, the anti-BCMA antibodies described herein have an increased half-life (e.g., relative to a control, e.g., a reference antibody, e.g., an antibody that cross-competes for BCMA binding), and thus the anti-BCMA antibodies can be administered to individuals at increased intervals between doses. throw. For example, the anti-BCMA antibody can be administered weekly, every two weeks, every three weeks, every four weeks, every 6 weeks, every 8 weeks, or for longer periods of time.

In some embodiments, the therapeutically effective amount of an anti-BCMA antibody or fragment thereof described herein is about 90%, 80%, 70%, 60% of the effective amount of a reference therapeutic protein (e.g., an antibody that cross-competes for BCMA binding) , 50%, 40%, 30%, 20%, 10% or 5%. In some embodiments, a single dose of an anti-BCMA antibody described herein achieves a therapeutic effect comparable to two or more doses of a reference antibody.

In some embodiments, an anti-BCMA antibody or fragment thereof described herein is present in about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of individuals. The dose is administered at a concentration of the target antigen (e.g., BCMA).

In some embodiments, an anti-BCMA antibody or fragment thereof described herein can be physically introduced into an individual using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, Intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid space, intraspinal, epidural and sternum intravenous injection and infusion, and in vivo electroporation. In some embodiments, the formulations are administered via parenteral routes, including topical, epidermal, or mucosal routes of administration, such as intranasal, transvaginal, rectal, sublingual, or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended time periods.

In some embodiments, anti-BCMA antibodies or fragments thereof described herein can be used in a variety of diagnostic and therapeutic applications. For example, detectably labeled versions of engineered antibodies as described herein can be used in assays that detect the presence or amount of BCMA in a sample (eg, a biological sample). The engineered antibodies described herein can be used in in vitro assays to study binding to BCMA. In some embodiments, anti-BCMA antibodies described herein can be used as positive controls in assays designed to identify additional novel compounds that may additionally be used to treat BCMA-related disorders. For example, the anti-BCMA antibodies described herein can be used as positive controls in assays identifying additional compounds that bind to BCMA (eg, small molecules, aptamers, or antibodies).

The antibodies or antigen-binding fragments thereof described herein can be used to monitor individuals, such as those who have, are suspected of having, are at risk of developing, or are being treated for one or more BCMA-related disorders. Monitoring may include determining the amount or activity of BCMA in the individual, for example, in the individual's serum. In some embodiments, the assessment is at least one (1) hour, such as at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, Or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or longer. Individuals may be assessed at one or more of the following times: before treatment begins; during treatment; or after one or more treatment components have been administered. Assessment may include assessment of the need for further treatment, for example, assessment of whether the dosage, frequency of administration, or duration of treatment should be changed. Assessment may also include evaluating the addition or deletion of selected treatment modalities, such as the need to add or delete any treatment for BCMA-related conditions described herein. In one embodiment of the present invention, a chimeric antigen receptor (CAR) comprising a BCMA binding domain is disclosed for the treatment of various hematological malignancies including multiple myeloma. BCMA protein is expressed on cancer cells. The BCMA antigen-binding portion of the CAR interacts with epitopes within the extracellular domain of its BCMA fragment.

In certain embodiments, CAR constructs, nucleic acid sequences, vectors, immune cells, etc., as contemplated herein, and/or pharmaceutical compositions containing the same, are used to prevent, treat, or ameliorate disease, such as cancerous disease. In certain embodiments, for example, pharmaceutical compositions of the present disclosure may be particularly useful in preventing, ameliorating, and/or treating cancer, including cancers that express specific antigens and may or may not be solid tumors.

In particular embodiments, the immune cells utilizing the receptors may be NK cells, T cells, γδ T cells, αβ T cells, or NKT or invariant NKT (iNKT), or may be engineered to perform cell therapy in mammals. Transform NKT cells. In the case of cell lines such as NK cells, the NK cell therapy can be of any type and the NK cells can be of any type. In certain embodiments, cell lines are engineered to express NK cells that express one or more CARs and/or one or more suicide genes and/or one or more interleukins. In specific embodiments, the cell line is CAR-transduced NK cells.

In certain embodiments, the present disclosure contemplates that the disclosure may be administered alone or in any combination using standard vectors and/or gene delivery systems, and in at least some aspects, together with pharmaceutically acceptable carriers or excipients. Provided CAR expressing cells, CAR constructs, CAR nucleic acid molecules and CAR vectors. In certain embodiments, following administration, the nucleic acid molecule or vector can be stably integrated into the genome of the individual. middle.

In certain embodiments, viral vectors that are specific for certain cells or tissues and persist in NK cells may be used. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared in accordance with the present disclosure may be used to prevent or treat or delay the diseases identified above.

In addition, the present disclosure relates to methods for preventing, treating, or ameliorating neoplastic diseases, which methods include the step of administering to an individual in need thereof an effective amount of cells expressing a CAR, a nucleic acid sequence, or a vector, as contemplated herein. and/or produced by methods as contemplated herein.

Possible indications for the administration of compositions of exemplary CAR cells are cancerous diseases, including neoplastic diseases, including, for example, B-cell malignancies, multiple myeloma, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer. . Exemplary indications for administering compositions of antigen-targeting CAR cells are cancerous diseases, including any malignancy that expresses the antigen. Administration of the compositions of the present disclosure may be used in all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early-stage cancer, late-stage cancer, and/or metastatic cancer and/or refractory cancer.

The present disclosure further contemplates co-administration regimens with other compounds that act via immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical regimens for co-administration of the compounds of the present invention may encompass co-administration at the same time, before or after the administration of the other components. Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

In a specific aspect, the present invention provides a method of inhibiting the proliferation of a BCMA-expressing cancer cell population or reducing the BCMA-expressing cancer cell population, the method comprising combining the BCMA-expressing cancer cell population with the BCMA-expressing cells of the present invention. Anti-BCMA CAR expressing cells (eg, BCMA CAR expressing NK cells) are contacted. In one aspect, the present invention provides a method for inhibiting the proliferation of a BCMA-expressing cancer cell population or reducing the BCMA-expressing cancer cell population, the method comprising causing A population of BCMA cancer cells is contacted with an anti-BCMA CAR-expressing cell of the invention (eg, a BCMA CAR-expressing NK cell) that binds to BCMA-expressing cells. In certain aspects, anti-BCMA CAR-expressing cells (e.g., BCMA CAR-expressing NK cells) of the invention reduce the risk of myeloid leukemia or another cancer associated with BCMA-expressing cells relative to a negative control in an individual or animal model. The number, amount or percentage of cells and/or cancer cells is at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95% or at least 99%. In one aspect, the individual system person.

In some embodiments, cancers for which the present treatment methods are useful include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors may include, but are not limited to, organ tumors selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and mammary gland. Exemplary hematological tumors include myeloid tumors, T or B cell malignancies, leukemias, lymphomas, blastomas, myeloma, and the like. Other examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric/stomach cancer ( Including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (kidney /renal cancer), prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

Cancer may specifically be of the following histological types, but is not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma ; Basal cell carcinoma; Trichoblastic carcinoma; Transitional cell carcinoma; Papillary transitional cell carcinoma; Adenocarcinoma; Malignant gastrinoma; Cholangiocarcinoma; Hepatocellular carcinoma; Combined hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenocarcinoma; Adenocarcinoma cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli adenocarcinoma; parenchymal carcinoma; malignant carcinoid; branchial-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; oncocytic carcinoma; eosinophilic adenocarcinoma; basophilic Carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; noncapsular sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine carcinoma; sebaceous glands Carcinoma; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant coma; Malignant granulosa cell tumor; malignant fibroblastoma; Sertoli cell carcinoma; malignant stromal cell tumor; malignant lipocytoma; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angiosarcoma; malignant melanoma; colorless Pigmented melanoma; superficial spreading melanoma; lentiginous melanoma; acral melanoma; nodular melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma ; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Interstitial sarcoma; Malignant mixed tumor; Mullerian mixed tumor; Wilms tumor; Hepatoblastoma Cell tumor; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian stroma; villous cell carcinoma Membranous carcinoma; malignant mesonephrosoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal Chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumors; Ameloblast odontal sarcoma; Malignant ameloblastoma; Ameloblast fibrosarcoma; Malignant pineal tumor; Chordoma; Malignant glioma ; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioblastoma; Primitive extraneural Germ layer; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; malignant meningiomas; neurofibrosarcomas; malignant schwannomas; malignant granulosa cell tumors; malignant lymphomas; Hodgkin's disease; Hodgkin's disease; Hodgkin's disease Bud tumors; small lymphocytic malignant lymphoma; large cell diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular lymphoma Non-Hodgkin lymphoma (NHL); small lymphocyte (SL) NHL; intermediate-grade/follicular non-Hodgkin lymphoma; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastoid NHL; high-grade small nonlytic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative Small intestinal diseases; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid Sarcomas; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myelogenous leukemia.

As non-limiting examples, the anti-BCMA antibodies, CARs, cells and compositions of the invention are used to treat B cell-related diseases. In some embodiments, the cancer is a B cell related disease. In some embodiments, the B cell-related disease is selected from the group consisting of: plasmacytoma, Hodgkin lymphoma, follicular lymphoma, small non-lytic nuclear cell lymphoma, endemic Burkitt lymphoma, sporadic Burkitt lymphoma, marginal zone lymphoma, extranodal mucosal lymphoma tissue lymphoma, nodular monocytic B-cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma neoplasm, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, pulmonary B-cell angiocentric lymphoma, small lymphocytic lymphoma, unknown malignant B-cell, lymphomatoid granuloma, post-transplant lymphoproliferative Disorders, immunoregulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's disease syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, rapidly progressive glomerulonephritis, Heavy chain disease and primary or immune cell amyloidosis.

Pharmaceutical composition

The pharmaceutical compositions of the present disclosure comprise an effective amount of a composition comprising NK cells dispersed in a pharmaceutically acceptable carrier. In some embodiments, the compositions include pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, and/or adjuvants. In some embodiments, the compositions include excipients. The preparation of pharmaceutical compositions containing such compositions in light of the present disclosure will be known to those skilled in the art, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Edition Lippincott Williams and Wilkins, 2005, which Incorporated herein by reference. Additionally, for administration to animals (e.g., humans), it is understood that preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, Absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes and other materials and their combinations, such as those of general craftsmen Known (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1289-1329, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

A pharmaceutical composition may contain different types of carriers depending on whether it is administered in solid, liquid or aerosol form and whether sterility is required for such route of administration such as injection. The composition disclosed in the present invention can be intravenous, intradermal, transdermal, intrathecal, intraarterial, endoscopic, intraperitoneal, intranasal, intravaginal, intrarectal, topical, intramuscular, transdermal, subcutaneous, mucosal , oral, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct local infusion irrigation Target cells, via a catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), by local perfusion, in the tumor microenvironment, or by other methods as known to those of ordinary skill or any combination of the foregoing (see, eg, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which is incorporated herein by reference).

Preparation and administration

In various embodiments, an antibody described herein, or an antigen-binding fragment thereof (eg, an anti-BCMA antibody described herein) can be incorporated into a pharmaceutical composition. Such pharmaceutical compositions may be used, for example, to prevent and/or treat diseases, such as BCMA-related disorders. Pharmaceutical compositions may be formulated by methods known to those skilled in the art (eg, as described in Remington's Pharmaceutical Sciences , 17th ed., editor Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985)).

The appropriate injection method can be selected according to the age and condition of the individual. A single dose of a pharmaceutical composition containing an antibody described herein or a fragment thereof (eg, an anti-BCMA antibody described herein) can be selected from the range of 0.001 to 1000 mg/kg body weight. On the other hand, the dosage may be selected in the range of 0.001 to 100000 mg/body weight, but the present disclosure is not limited to such ranges. The dosage and method of administration vary depending on the patient's weight, age, condition and the like, and can be appropriately selected as necessary by a person skilled in the art.

In various embodiments, pharmaceutical compositions may be formulated to include pharmaceutically acceptable carriers or excipients. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions of the present invention may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

In various embodiments, compositions, such as sterile injectable formulations, including antibodies as described herein, may be formulated in accordance with common pharmaceutical practice using distilled water for injection as the vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as aqueous solutions for injection, combined with appropriate solubilizers as appropriate. Used are, for example, alcohols such as ethanol and polyols such as propylene glycol or polyethylene glycol, and non-ionic surfactants such as polysorbate 80 ^™ , HCO-50 and the like.

As disclosed herein, pharmaceutical compositions may be in any form known in the art. Such forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

The selection or use of any particular form may depend in part on the intended mode of administration and therapeutic application. For example, compositions containing compositions for systemic or local delivery may be in the form of injectable or infusible solutions. Accordingly, the compositions may be formulated for administration by parenteral modes (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, intrapulmonary, intramuscular, intraarterial Intrathecal, intrathecal, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid space, intraspinal, epidural, Intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

The route of administration may be parenteral, such as by injection, nasal administration, transpulmonary administration, or transdermal administration. Systemic or local administration can be carried out by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

In various embodiments, the pharmaceutical compositions of the present invention can be formulated into solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of a composition described herein in an appropriate solvent with one or a combination of ingredients enumerated above, as appropriate, followed by filtered sterilization. Typically, dispersions are prepared by The compositions are prepared by incorporating a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield powders of the compositions described herein plus any other desired ingredients from their previously sterile-filtered solutions (see below). Proper flowability of the solution can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

Pharmaceutical compositions may be administered parenterally in the form of injectable formulations comprising sterile solutions or suspensions in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions may be formulated by appropriately combining the therapeutic molecule with a pharmaceutically acceptable vehicle or medium such as sterile water and physiological saline, vegetable oils, emulsifiers, suspending agents, interfaces, etc. Active agents, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders and then mixed into unit dosage forms required by generally accepted pharmaceutical practice. The active ingredient is included in the pharmaceutical preparation in such an amount as to provide a suitable dosage within the specified range. Non-limiting examples of oily liquids include sesame oil and soybean oil, and this can be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injection may be packaged in suitable ampoules.

In some embodiments, the formulated composition may be frozen for storage at a temperature below 0°C (eg, -20°C or -80°C). Cryopreserved cells are thawed and injected into individuals. As culture media for freezing formulations, the following cryopreservation media are exemplified;

The ingredients for frozen formulations can be obtained from any commercial source. As non-limiting examples, each of the above components is available from the following suppliers:

In some embodiments, the composition can be formulated for storage at 2-8°C (e.g., 4°C) for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1.5 years or 2 years). Thus, in some embodiments, compositions described herein are stable when stored at 2-8°C (eg, 4°C) for at least 1 year.

In certain cases, pharmaceutical compositions may be formulated as solutions. In some embodiments, the composition may, for example, be formulated as a buffered solution at a suitable concentration and suitable for storage at 2-8°C (eg, 4°C).

Compositions including one or more engineered antibodies as described herein can be formulated in immunoliposome compositions. Such formulations may be prepared by methods known in the art. Liposomes with extended circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

In certain embodiments, the compositions can be formulated with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are known in the art. See, for example, JR Robinson (1978) " Sustained and Controlled Release Drug Delivery Systems ," Marcel Dekker, Inc., New York.

In some embodiments, the compositions may be formulated to be suitable for intrapulmonary administration (eg, via an inhaler or nebulizer) to a mammal, such as a human. Methods of formulating such compositions are well known in the art. Dry powder inhaler formulations and suitable systems for administering such formulations are also known in the art. Pulmonary administration can be oral and/or nasal. Examples of drug devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPI), and nebulizers. For example, the compositions described herein can be administered to the lungs of an individual via a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the medical field and include, but are not limited to: TURBOHALER® (AstraZeneca; London, England), AIR® inhaler (ALKERMES®; Cambridge, Mass.); ROTAHALER® (GlaxoSmithKline; London, England); and ECLIPSE ^™ (Sanofi-Aventis; Paris, France). See also, for example, PCT Publications Nos. WO 04/026380, WO 04/024156 and WO 01/78693. DPI devices have been used for pulmonary delivery of peptides such as insulin and growth hormone. In some embodiments, compositions described herein can be administered intrapulmonary via a metered dose inhaler. These inhalers rely on propellants to deliver discrete doses of a compound to the lungs. Other devices and methods of intrapulmonary administration are described, for example, in U.S. Patent Application Publication Nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, the formulated compositions are delivered to the eye, for example, in the form of a pharmaceutically acceptable solution, suspension, or ointment. Preparations for treating the eye may be in the form of sterile aqueous solutions Formulas containing, for example, additional ingredients such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, and tackifiers. Formulations as described herein may be administered topically to an individual in need of treatment by conventional methods, such as in the form of drops or by dipping the eye in a therapeutic solution containing one or more compositions (e.g., eyes of individuals with AMD).

In certain embodiments, a variety of devices for introducing drugs into the vitreous cavity of the eye may be suitable for administration of compositions as described herein. For example, US Publication No. 2002/0026176 describes a drug-containing plug that can be inserted through the sclera so that it extends into the vitreous cavity to deliver pharmaceutical agents into the vitreous cavity. In another example, US Patent No. 5,443,505 describes an implantable device for introduction into the suprachoroidal space or avascular area for sustained release of drugs into the interior of the eye. U.S. Patent Nos. 5,773,019 and 6,001,386 each disclose an implantable drug delivery device that can be attached to the scleral surface of the eye. Other methods and devices for delivering therapeutic agents to the eye (eg, transscleral patches and delivery via contact lenses) are described, for example, in Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11): 1164-1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1): 1-10(10); Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45: 2342-2347; Kim et al. (2007) Ophthalmic Res 39:244-254; and PCT Publication No. WO04/073551, the disclosures of which are incorporated herein by reference in their entirety.

In certain embodiments, administration of an antibody as described herein is accomplished by administering to an individual a nucleic acid encoding the antibody. Nucleic acids encoding therapeutic antibodies described herein can be incorporated into genetic constructs for use as part of a gene therapy regimen to deliver nucleic acids that can be used to express and produce antibodies within cells. Expression constructs for such components may be administered in any therapeutically effective vehicle, such as any formulation or composition capable of effective gene delivery of the component to cells in vivo . Methods include inserting the test gene into a viral vector, including recombinant retrovirus, adenovirus, adeno-associated virus, lentivirus and herpes simplex virus-1 (HSV-1), or recombinant bacteria or eukaryotic plasmids. Viral vectors can directly transfect cells; plastid DNA can be transfected with the help of, for example, cationic liposomes (lipofectin) or derivatized polylysine conjugates, gramicidin S, artificial virus envelopes or other such intracellular carriers, and delivery by direct injection of genetic constructs or CaPO ₄ precipitates (see, eg, WO04/060407). Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM known to those skilled in the art (see, e.g., Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85: 6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA 85: 3014-3018; Armentano et al. (1990) Proc Natl Acad Sci USA 87: 6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88: 8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895; Hwu et al. (1993) J Immunol 150:4104 -4115; U.S. Patent Nos. 4,868,116 and 4,980,286; and PCT Publications Nos. WO89/07136, WO89/02468, WO89/05345 and WO92/07573). Another viral gene delivery system utilizes adenovirus-derived vectors (see, e.g., Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68 :143-155). Suitable adenoviral vector systems derived from the adenovirus Ad type 5 dl324 strain or other adenovirus strains (eg, Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Another viral vector system that can be used to deliver test genes is adeno-associated virus (AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356; Samulski et al. (1989) J Virol 63:3822-3828; and McLaughlin et al. (1989) J Virol 62:1963-1973 .

In various embodiments, subcutaneous administration may be by means of, for example, a syringe, a prefilled syringe, an autoinjector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable syringe, a subcutaneous The infusion unit is equipped with a mobile syringe infusion pump or other devices that are combined with subcutaneously injected antibody drugs.

The injection system of the present disclosure may employ a delivery pen as described in US Pat. No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to diabetic patients, are well known in the art. Such devices may include at least one injection needle (e.g., a 31 gauge needle approximately 5 mm to 8 mm in length), typically prefilled with one or more therapeutic unit doses of a therapeutic solution, and may be used to rapidly deliver the solution to an individual with as little pain as possible . A drug delivery pen includes a vial holder in which a vial of a therapeutic or other drug may be received. The pen may be a completely mechanical device, or it may be integrated with electronic circuitry to accurately set and/or indicate the dosage of medication injected into the user's body. See, for example, U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the set includes one or more disposable replacement needles. Pen devices suitable for delivering any of the compositions featured in this invention are also described in, for example, U.S. Patent Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described, for example, in U.S. Patent No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the precision pen injector (PPI) device MOLLY ^™ manufactured by Scandinavian Health Ltd.

In some embodiments, the compositions described herein can be therapeutically delivered to an individual by topical administration. As used herein, "local administration" or "local delivery" may refer to delivery that does not rely on transport of a composition or agent through the vascular system to its intended target tissue or site. For example, the composition may be delivered by injecting or implanting the composition or agent or by injecting or implanting a device containing the composition or agent. In certain embodiments, upon local administration near a target tissue or site, the composition or agent, or one or more components thereof, can diffuse to Intended target tissue or site other than the site of administration.

In some embodiments, compositions provided herein are presented in unit dosage form, which unit dosage form may be suitable for self-administration. Such unit dosage forms may be provided in containers, typically such as vials, cartridges, prefilled syringes or disposable pens. Metering meters, such as the metering device described in US Pat. No. 6,302,855, may also be used, for example, with the injection systems described herein.

Suitable dosages of the compositions described herein that are capable of treating or preventing a disorder in an individual will depend on a variety of factors, including, for example, the age, sex, and weight of the individual to be treated and the particular inhibitor compound used. For example, treatment of an individual suffering from a BCMA-related disorder may require different dosages of a composition including the antibody as compared to dosages of different formulations of the antibody as described herein. Other factors that affect the dosage administered to an individual include, for example, the type or severity of the condition. For example, an individual with one BCMA-related condition may require different doses than an individual with another BCMA-related condition. Other factors may include, for example, other medical conditions affecting the individual concurrently or previously, the individual's general health, the individual's genetic predispositions, diet, timing of administration, excretion rate, combination of medications, and any other therapeutic agents administered to the individual. . It is also understood that the specific dosage and treatment regimen for any particular individual may be adjusted based on the judgment of the treating physician.

In some embodiments, compositions described herein are administered in fixed doses or in milligrams per kilogram (mg/kg) doses. In some embodiments, the dosage is selected to reduce or avoid the production of antibodies or other host immune responses against one or more antibodies or antigen-binding fragments thereof in the composition. Although in no way intended to be limiting, exemplary dosages for antibodies such as compositions described herein include, for example, 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg , 1-20mg/kg and 1-10mg/kg. Exemplary dosages of compositions described herein include, but are not limited to, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg.

In some embodiments, the composition is administered at a dose and at a dosing interval sufficient to alleviate or treat one or more symptoms of the disease associated with abnormal BCMA manifestations. In some embodiments, the administration is daily, twice daily, once a week, twice a week, three times a week, once every two weeks, once a month, once every 2 months, once every 6 months, or once a year. composition. In some embodiments, treatment involves a single administration of the antibody.

In some embodiments, the composition is a pharmaceutical composition including one or more excipients.

Pharmaceutical solutions may include a therapeutically effective amount of a composition described herein. Such effective amounts can be readily determined by one of ordinary skill in the art based in part on the effect of the composition administered or the effect of the composition in combination with one or more additional active agents if more than one agent is used. The therapeutically effective amount of a composition described herein may also vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the composition (and one or more additional active agents) to elicit the desired response in the individual. This needs to reflect, for example, an improvement in at least one condition parameter, such as an improvement in at least one symptom of a BCMA-related disorder. For example, a therapeutically effective amount of a composition described herein may inhibit (reduce the severity or eliminate the occurrence of) and/or prevent a particular disorder and/or any of the particular disorders known in the art or described herein. Symptoms. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

Suitable human doses of any composition described herein can be further evaluated, for example, in a Phase I dose escalation study. See, e.g., vanGurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1)q5q3-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

The toxicity and therapeutic efficacy of the composition can be determined by known pharmaceutical procedures in cell cultures or experimental animals (eg, animal models of any BCMA-related disorders). Such procedures can be used, for example, to determine LD ₅₀ (lethal dose for 50% of the population) and ED ₅₀ (therapeutic effective dose for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio _LD50 / _ED50 . Compositions described herein that exhibit a high therapeutic index are preferred. Although compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of the affected tissue and minimizes potential damage to normal cells, thereby reducing side effects.

Those skilled in the art will understand that data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for use in humans. Appropriate dosages of the compositions described herein are generally within a range of circulating concentrations of the composition that includes the ED ₅₀ and has little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For the compositions described herein, the therapeutically effective dose can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations that includes _I0 (i.e., the antibody concentration that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. For example, levels in plasma can be measured by high performance liquid chromatography. In some embodiments, for example, where local administration is desired (eg, to the eye or joint), cell culture or animal modeling can be used to determine the dosage required to achieve a therapeutically effective concentration within the local site.

combination ratio method

In accordance with the present disclosure, antibodies, antigen-binding fragments, CARs, CAR-expressing vectors and cells, and compositions described herein may be used in combination with other therapies for the treatment of cancer.

In various embodiments, an anti-BCMA antibody as described herein can be included in a treatment course further comprising administering to the individual at least one additional agent. In various embodiments, the additional agent administered in combination with an anti-BCMA antibody as described herein can be a chemotherapeutic agent.

In some embodiments, anti-BCMA antibodies, fragments thereof, can be conjugated to (e.g., linked to) therapeutic agents (e.g., chemotherapeutic agents and radioactive atoms) for binding to cancer cells to deliver the therapeutic agents to cancer cells and kill cancer cells expressing human BCMA. In some embodiments, anti-BCMA antibodies are linked to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, an interleukin, a radioactive atom, a siRNA, or a toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the agent is a radioactive atom.

In some embodiments, these methods can be performed in conjunction with other therapies for BCMA-related conditions. For example, the composition can be administered to an individual simultaneously with, before, or after chemotherapy. In some embodiments, the composition can be administered to an individual simultaneously with, before, or after an adoptive treatment method.

In various embodiments, additional doses administered in combination with an anti-BCMA antibody as described herein can be administered at the same time as the anti-BCMA antibody, on the same day as the anti-BCMA antibody, or in the same week as the anti-BCMA antibody. give. In various embodiments, additional agents administered in combination with an anti-BCMA antibody as described herein can be administered in a single formulation with the anti-BCMA antibody. In certain embodiments, the additional dose is administered in a manner that is temporally separate from the administration of the anti-BCMA antibody as described herein, e.g., one or more hours, before or after the administration of the anti-BCMA antibody. One or more days, one or more weeks before or after, or one or more months before or after. In various embodiments, the frequency of administration of the one or more additional agents can be the same, similar, or different than the frequency of administration of the anti-BCMA antibodies as described herein.

Included in combination therapy are treatment regimens that include the administration of two different antibodies as described herein and/or treatment regimens that include administration of an antibody as described herein through multiple formulations and/or routes of administration.

In some embodiments, the compositions may be formulated with one or more additional therapeutic agents, such as additional therapies for treating or preventing BCMA-related disorders (eg, cancer or autoimmune disorders) in an individual. Additional agents used to treat BCMA-related conditions in an individual may vary depending on the specific condition being treated, but may include, but are not limited to, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, Ifosfamide (osfamide), carboplatin, etoposide, dexamethasone, cytarabine, cisplatin, cyclophosphamide amine or fludarabine.

The compositions described herein may replace or augment previously or currently administered therapies. For example, following treatment with a composition described herein, administration of one or more additional active agents can be discontinued or reduced, e.g., administered at a lower level, e.g., cross-over after administration of an anti-BCMA antibody described herein is reduced. Level of reference antibody competing for BCMA binding. In some embodiments, administration of previous therapy can be maintained. In some embodiments, prior therapy is maintained until the level of the composition reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

In certain embodiments, the compositions and methods of this embodiment involve a combination of a population of immune cells, including a population of NK cells, and at least one additional therapy. Additional therapies may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy , hormone therapy, oncolytic viruses, or a combination of the above. Additional therapy may be in the form of adjuvant or neoadjuvant therapy.

In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiting agent (eg, an agent intended to reduce the occurrence and/or severity of side effects of treatment, such as an anti-nausea agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. Additional therapy may be one or more chemotherapeutic agents known in the art.

In certain embodiments, in addition to the inventive cell therapies of the present disclosure, an individual may have been, could be, or will be provided with certain additional therapies for cancer, including surgery, radiation, immunotherapy One or more of (except cell therapy in this disclosure), hormone therapy, gene therapy, chemotherapy, etc.

The immune cell therapy can be administered before, during, after, or in various combinations relative to additional cancer therapy. The time interval between administrations can range from simultaneous to minutes to days to weeks. In embodiments where the immune cell therapy and the additional therapeutic agent are provided separately to the patient, one typically ensures that there is no long period between the times of each delivery such that the two compounds are still able to work in combination to the patient's benefit. In such cases, it is expected that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12 to 24 or 72 hours of each other, and more specifically, within about 6-12 hours of each other. In some cases, a significantly longer treatment period may be required, with anywhere from days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse.

Various combinations are possible. For the following example, the immune cell therapy is "A" and the anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Given the toxicity of the agent, if any, administration of any compound or cell therapy of this example to a patient will follow general protocols for the administration of such compounds. Thus, in some embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

A.Chemotherapy

According to this embodiment, a variety of chemotherapeutic agents may be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. Such agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, one can cross-link DNA directly, embed DNA or by Agents characterized by their ability to affect nucleic acid synthesis and induce chromosomal and mitotic aberrations.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, endosulfan and piposulfan; aziridines such as benzodiopa , carboquinone, madopa and udopa; ethyleneimines and methylmelamines, including melamine, triethylenemelamine, triethylenephosphatamide, triethylenethiophosphoramide and trimethylolmelamine; Annonaceae (especially bulatacin and bulatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; calistatin; CC-1065 (including its adodoxin Synthetic analogues of adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; Docamycin (including synthetic analogues KW-2189 and CB1-TM1); eleutheroine; panlastatin; botulinum toxin; spongostatin; nitrogen mustards, such as chlorambucil and chlorambucil mustard, chlorphosphonamide, estramustine, ifosfamide, ethyl chloride, ethyl chloride hydrochloride, melphalan, novibicin, fenestine, prednimustine, tricholine Phosphatide and uracil mustard; nitrosoureas, such as carmustine, chlorozolin, flutimustine, lomustine, nimustine and ranistine; antibiotics, such as Enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin ω1I); danimycin, including danimycin A; bisphosphonates, such as clodronate salt; espamycin; and new carcinogen chromophores and related chromoprotein enediyne antibiotic chromophores, aclarithromycin, actinomycin, anthromycin (authrarnycin), diazoserine , bleomycin, actinomycin, carbomycin, carminin, carcinin, chromomycin, actinomycin, daunorubicin, detobicin, 6-diaza-5-side Oxy-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin (bicin), epirubicin, esopubicin, idarubicin, marseicin, mitomycins such as mitomycin C, mycophenolic acid, nogaramycin, olivinemycin, pero Mycin, pofimycin, puromycin, quiramycin, rhodobicin, streptozotocin, streptozocin, tuberculin, ubenimex, ipinostatin and zorubicin ; Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, deteroxine, and trimethate; purine analogs, such as fludarabine, 6-mercaptopurine , thiamine purine and thioguanine; pyrimidine analogues such as amcitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, dosifluridine , excitabine and floxuridine; androgens, such as carrutestone, drostandrosterone propionate, thiandrostenol, mestandroane and testosterone; anti-adrenergics, such as mitotane and trilostane ; Folic acid supplements, such as leucovorin; Acetyl Acetone; Aldehyde Phosphate Glycoside; Aminoacetate; Eniluracil; Acridine; Phenylbutyrate; Bisantrene; Edatroxate; Metephedrine Bases; norgoxin; diazinon; metformin; erititonium acetate; epothilone; gastrin; gallium nitrate; hydroxyurea; lentinan; lonidanin; maytansinoids, such as maytansine And ansithromycin; mitoguanine; mitoxantrone; mopidamole; nitroaniline; pentostatin; methamine; pirarubicin; loxantrone; podophylline acid; 2- Ethylhydrazine; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizobium; Cizofran; Spirogermanium; Myosinic acid; Triazone; 2,2',2"-Trichlorotriethylamine ; Trichothecenes (especially T-2 toxin, veraculin A, lorridin A and serpentine); polyurethanes; vindesine; dacarbazine; mannomustine; mitobromidol; rice Trolide; piperobromide; cytosine; arabinoside ("Ara-C"); cyclophosphamide; paclitaxel, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum complex Complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; Novanlone; teniposide; idatrexate; daunorubicin; aminopterin; Xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor Agent RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, primycin, gemcitabine, navibine, farnes protein transferase inhibitors, transplatin and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

B. Radiotherapy

Other factors that cause DNA damage and are widely used include the targeted delivery of what are commonly called gamma rays, X-rays, and/or radioactive isotopes to tumor cells. Other forms of DNA damage, such as microwave, proton beam (US Patents 5,760,395 and 4,870,287) irradiation, and UV irradiation, were also considered. It is likely that all of these factors cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens per day for an extended period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation, and uptake by tumor cells.

C.Immunotherapy

Those skilled in the art will understand that additional immunotherapies may be combined or used in combination with the methods of the examples. In the case of cancer treatment, immunotherapy often relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example of this. Immune effectors may be, for example, antibodies specific for certain markers on the surface of tumor cells. The antibody can act alone as an effector of the therapy, or it can recruit other cells to actually effect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used as targeting agents. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

Antibody-drug conjugates have become a breakthrough approach in developing cancer therapies. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for their antigenic targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing Low toxicity and improved therapeutic index. Two ADC drugs approved by the FDA, ADCETRIS® (brentuximab vidotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, have validated this approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become more sophisticated, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulation/high level expression in tumor cells and robust internalization.

In one aspect of immunotherapy, tumor cells must carry some easily targetable markers that are not present on most other cells. There are many tumor markers and any of these may be suitable for targeting in the context of this example. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy combines anticancer effects with immune stimulation. Immunostimulatory molecules are also present, including: interleukins, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, and chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand.

Examples of immunotherapies currently being studied or used are immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (US Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al. (Man, 1998); interleukin therapy, such as interferon alpha, beta and gamma, IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy , such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up the signal (e.g., costimulatory molecules) or turn it down. Suppressive immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T Lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), program Chemical death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3) and V domain Ig inhibitor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

Immune checkpoint inhibitors can be drugs, such as small molecules, ligands or recombinant forms of receptors, or specifically antibodies, such as human antibodies (eg, International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4) ): 252-64, 2012; both are incorporated herein by reference). Inhibitors of known immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized or human forms of antibodies may be used. As will be appreciated by those skilled in the art, alternative and/or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and/or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binds to the partner species PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific form, PDL1 binds to the partner species PD-1 and/or B7-1. In another embodiment, PDL2 binding antagonists are molecules that inhibit the binding of PDL2 to its binding partners. In a specific form, PDL2 binds to the partner species PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide. Exemplary antibodies are described in US Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as are described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference. .

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). adhesin). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.

Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is present on the surface of T cells and when combined with antigen-presenting cells CD80 or CD86 on the cell surface acts as an "off" switch when bound. CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T-cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and may be important for their function. Activation of T cells via T cell receptors and CD28 results in increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies may be used. For example, anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as Qumei Tremelimumab; formerly known as tremelimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004 ) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58: 5301-5304. The teachings of each of the above-mentioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; all of which are incorporated herein by reference.

An exemplary anti-CTLA-4 antibody system is ipilimumab (also known as 10D1, MDX-010, MDX-101 and Yervoy®) or antigen-binding fragments and variants thereof (see, eg, WO 01/14424). In other embodiments, the antibody comprises the heavy chain and light chain CDRs or VR of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding or binding to the same epitope on CTLA-4 as the antibody described above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). zone consistency).

Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated by reference. are incorporated herein by reference, and immunoadhesins such as those described in U.S. Patent No. 8,329,867 are incorporated herein by reference.

D.Surgery

Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, resected, and/or destroyed, and may be used in conjunction with other therapies such as the treatment of this example, chemotherapy, radiation therapy, hormonal therapy , gene therapy, immunotherapy and/or alternative therapy. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs surgery).

When all or part of a cancer cell, tissue or tumor is removed, a cavity may form in the body. Treatment can be accomplished by infusion, direct injection, or topical application of additional anti-cancer therapies to the area. Such treatments may be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks or Every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also have different dosages.

E. Recombinant gene technology

According to the present disclosure, conventional molecular biology, microbiology and recombinant DNA technologies within the technical scope of this technology can be used. Such techniques are described in the literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach , Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (edited by MJGait 1984); Nucleic Acid Hybridization (edited by BDHames & SJHiggins (1985)); Transcription And Translation (edited by BDHames & SJHiggins (1984)); Animal Cell Culture ( RIFreshney editor (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B.Perbal, A Practical Guide To Molecular Cloning (1984); FMAusubel et al (editor), Current Protocols in Molecular Biology , John Wiley & Sons, Inc. (1994).

Recombinant expression of a gene, such as a nucleic acid encoding a polypeptide such as an anti-BCMA antibody described herein, may involve construction of an expression vector containing a nucleic acid encoding the polypeptide. In some embodiments, the BCMA nucleic acid sequence can be a codon-optimized sequence for expression in mammalian cells. Once the polynucleotide is obtained, vectors for producing the polypeptide can be generated by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcription and translation control signals. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination.

Expression vectors can be transferred into host cells by conventional techniques, and the transfected cells can then be grown to produce polypeptides by conventional techniques. All publications, patent applications mentioned in this article, Patents and other references are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

F.Other agents

It is contemplated that other agents may be used in combination with certain aspects of this embodiment to enhance the efficacy of the treatment. Such additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers, or other biological agents. . Increasing intercellular signaling by increasing the number of GAP connections will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may be used in combination with certain aspects of this embodiment to enhance the anti-hyperproliferative efficacy of the treatment. Consider using cell adhesion inhibitors to improve the efficacy of this example. Examples of cell adhesion inhibitors are local adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of this embodiment to enhance therapeutic efficacy.

The set of this disclosure case

Any composition described herein may be included in the kit. In one non-limiting example, cells, reagents for producing cells, vectors, and reagents for producing vectors and/or components thereof may be included in a kit. In certain embodiments, NK cells may be included in the panel and they may or may not yet express a BCMA-CAR comprising (a) a CD28 hinge, an optional interleukin, or an optional suicide gene. Such a kit may or may not have one or more reagents for manipulating cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and/or their combination. Nucleotides encoding one or more CARs, suicide gene products, and/or interleukins may be included in the set. Proteins, such as interleukins or antibodies, including monoclonal antibodies, may be included in the kit. Nucleotides encoding engineered CAR receptor components are included in the kit, including reagents that produce the same components.

Example

The following examples are included to demonstrate certain embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention, and therefore may be considered to constitute certain modes of its practice. However, those skilled in the art should, in light of this disclosure, understand that many changes could be made in the specific embodiments disclosed and still obtain a similar or similar result without departing from the spirit and scope of the invention.

Example 1: Generation and conjugation of anti-BCMA monoclonal antibodies

This example demonstrates the characterization of anti-BCMA antibodies. Mice (Trianni mice carrying human transgenic heavy chain locus and kappa locus, 8-10 weeks old) were immunized according to the method described in the literature. For primary immunization, animals were injected into the hocks with 5 μg of recombinant BCMA-mouse Fc as an emulsion and an equal volume of GerbUMM. Mice were immunized in the same manner every 3-4 days for a total of 10 booster immunizations. Animals with high antigen-specific antibody titers were pre-fusion boosted with the same immunogen four days before spleen and lymph node harvest for further processing for RNA extraction or hybridoma fusion.

Spleens were isolated from mice and transferred to 50 ml tubes containing 10 ml RPMI, 10% FBS (0.2um filtered) and physically separated to isolate lymphocytes. Lymphocytes were harvested by centrifugation at 1200 rpm for 10 minutes. Lymphocytes were suspended in EasySep buffer (2ml; 0.2um filtered).

Anti-BCMA scFvs were identified by generating and selecting immune scFv phage display libraries. First, total RNA was purified from a single cell suspension of spleen cells from an individual mouse previously immunized with BCMA-expressing CHO cells. Total RNA was then used as a template to generate cDNA using random primers. scFv libraries were generated in mice to amplify variable regions from mouse antibody genes. The heavy chain and kappa chain amplicons were combined and cloned into the phagemid vector. Three rounds of selection were performed on recombinant BCMA proteins (human BCMA-Fc, human BCMA-6His) and/or cells expressing cynomolgus BCMA to identify BCMA-specific scFv phage clones. Periplasmic extracts of selected scFv clones are then screened by ELISA or flow cytometry. Colonies were selected for sequencing and binding confirmed by Octet using periplasmic extracts. A total of 19 clones were identified via phage display. In addition to the 19 strains, 42 other newly identified BCMA antibody strains were also used for antibody selection. Table 2 shows the binding affinity of human BCMA scFv to human BCMA.

Octet analysis

Octet binding assays were performed to assess the kinetics of binding to BCMA.

Octet binding assays were performed using human BCMA proteins with a panel of anti-BCMA strains for binding characterization. Human BCMA is first captured onto an appropriately coated Octet sensor tip, which is then positioned into a well containing an anti-BCMA colony for association. After placing the sensor tip in the dissociation well, the response was calculated for the interaction of each anti-BCMA strain and the corresponding human BCMA protein.

FACS analysis

CHO cells expressing BCMA were used and cell binding of anti-BCMA strains was tested by FACS. Cells are incubated with anti-BCMA strains and subsequently with secondary antibodies capable of binding anti-BCMA strains. The samples were washed appropriately and evaluated on an Attune NxT flow cytometer (Thermo Fisher Scientific). FACS analysis was performed in FlowJo.

Table 3 shows the results of Octet and FACS analysis of exemplary anti-BCMA strains against exemplary anti-BCMA antibodies and human BCMA. Exemplary Fabs (IgG) were observed to have high k _on rates and low k _off rates, with _K values in the low picomole range.

Example 2: chimeric antigen receptor construct

This example shows an exemplary CAR construct for reducing tumor burden. Figure 1 shows a CAR construct containing a BCMA binding region.

BCMA CARs comprise an anti-BCMA specific binder, an IgG1 hinge or a CD28 hinge, a CD28 transmembrane domain, and an IL-15 interleukin (eg, soluble IL-15). Such CAR may comprise the amino acid of SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49.

Example 3: In vitro efficacy of BCMA IgG hinge and BCMA CD28 hinge CAR constructs

This example shows the efficacy of a BCMA CAR containing a CD28 hinge domain as SEQ ID NO 45 compared to an IgG hinge domain in CB-NK cells.

CB-NK cells were evaluated in a standard four-hour 51 chromium (51Cr) release assay. Cytotoxicity. Briefly, target cells were loaded with 51Cr, washed, and co-cultured for 4 hours with CB-NK cells transduced with different CAR constructs with different BCMA-specific scFv conjugates and then removed supernatant and measure the 51Cr level using a gamma counter.

BCMA conjugates were tested in CAR backbones containing either an IgG1 hinge or a CD28 hinge. These CARs were then transduced into CB-NK and tested for in vitro killing efficacy using a chromium release killing assay.

All constructs showed higher killing than untransduced CB-NK, with the construct containing the CD28 hinge showing slightly enhanced killing, especially at lower E:T ratios, as shown in Figure 2 Visible.

Example 4: Comparison of the CD28 hinge domain and the IgG1 hinge domain in reducing tumor burden in vivo.

This example illustrates the benefits of using a BCMA-CAR construct with a CD28 hinge to reduce tumor burden in mice.

10 million BCMA CAR-NK cells (approximately 70% CAR+ve) containing BCMAIg1, BCMAIg2, BCMA28-1 or BCMA28-2 were used in this experiment. Female NSG mice aged 10-12 weeks were irradiated whole body with 150 cGy 24 hours before tumor inoculation. MM.1S-ffluc-MDA cells were prepared in PBS suspension at a concentration of ^2.5x10 cells/ml and used to inoculate cells intravenously at 0.5x10 cells/animal. Bioluminescence images were taken 1 day before dosing and 6 days after tumor inoculation, and animals were randomly divided into groups of 4 or 5 animals based on total flux. Animals were dosed 0, 1 or 9 days after tumor inoculation. The relevant concentration of CAR NK cells was resuspended in PBS and transferred to a vivarium on ice in small batches to ensure timely infusion into the animals while maintaining the viability of the CAR NK cells. Bioluminescence images were performed weekly on Xenogen IVIS to monitor tumor progression. Body weights were measured three times per week, and clinical observations were performed to monitor for any signs of toxicity. Microsampling (blood collection via submandibular) was performed weekly for cell kinetic analysis to quantify in vivo CAR NK amplification by ddPCR or flow cytometric analysis. At humane or research endpoints, animals from studies of interest are necropsied to obtain various tissues for pathological evaluation.

For qPCR CK, genomic DNA (gDNA) was extracted and purified from mouse whole blood samples (50 μL) using the MagMAX ^TM DNA Multi-Sample Ultra 2.0 kit in the presence of RNase A. The purified gDNA was then analyzed using an optimized digital droplet PCR (ddPCR)-based double-stranded analysis method designed to simultaneously determine the CAR construct and the reference a-actin 1 (ACTA1) gene (human and mouse The number of copies of a single copy of a gene in a genome. Tumor progression was monitored and imaged every 7 days. Figure 3 shows the growth of BCMA CAR with IgG hinge compared to BCMA CAR with CD28 hinge domain, relative to untreated mice (tumor only) and mice treated with non-transduced NK cells (NT NK). Tumor growth in mice treated with BCMA-CAR-transduced umbilical cord blood-derived NK cells.

A lower tumor burden was observed in mice receiving BCMA-CD28 compared to tumor or NT NK alone. Mice receiving BCMA CD28 had better tumor control during the first 2 weeks before succumbing to non-tumor burden-related death compared to BCMA IgG. Check the lungs, liver and spleen for lymphocyte infiltration. Figure 8 shows exemplary histopathology slides comparing lungs from normal mice to lungs from mice treated with BCMA28-2 CAR-NK cells. All tissues were fixed in 10% buffered formalin by immersion and intratracheal instillation into the lungs. After complete fixation (24-48 hours), organs were trimmed and processed for paraffin embedding. Paraffin blocks were sectioned at 4-6 microns, and tissue sections were mounted on glass slides, stained with hematoxylin and eosin (H&E), and covered with coverslips.

Weight fluctuations are summarized in Figure 12 .

Pathology evaluations were performed by board certified (ECVP) veterinary pathologists. neoplastic lesions Scores are based on their presence in the examined organ. Nonneoplastic lesions were scored according to their severity and distribution. Histopathology results were captured in the Pristima® software system.

Lymphocyte infiltration into the lungs, liver, and spleen was observed and is thought to be a cause of non-tumor burden-related death, as seen in Figure 8 . However, reducing the dose to 1 million versus 3 million total NK cells significantly improved non-tumor burden-related deaths and increased survival, as can be seen in Figure 4 .

Example 5: Comparison of anti-tumor activity of co-administered BCMA-CAR in MM1s tumor mice

This example compares the anti-tumor activity of exemplary co-administered BCMA-CAR NK cells.

One million BCMA CAR-NK cells (approximately 70% CAR+ve), containing SEQ ID NO: 19 or 20, were co-administered with MM1s tumors in mice. Tumor progression was monitored and imaged every 6-7 days. Figure 5 shows a graphical representation of survival of mice treated with 1 M BCMA-NK cells containing BCMA28-1 or BCMA28-2. It was observed that mice co-administered BCMA-CAR with BCMA28-2 performed best compared to mice co-administered with CAR-NK cells containing BCMA28-1, with more than 50% reduction in tumor-related deaths. Mice survived on day 77.

Example 6: Delayed dosing regimen for BCMA-CAR-NK and BCMA-CAR-T

This example shows that BCMA28-1 works best compared to other BCMA-CARs tested, however, all CAR constructs provide robust anti-tumor activity.

CAR-T cell production :

To produce CAR-T, human leukocyte isolation products (leukopaks) were purchased from Stem Cell Technologies. PBMCs were isolated from fresh leukocytes using a Ficoll density gradient. The EasySep Human T Cell Isolation Kit from Stemcell Technologies is used to purify T cells, which are then activated/expanded using T Cell TransAct beads from Miltenyi Biotec and research-grade IL-2. On the first day of T cell activation On day 2, cells were transduced with VSVG virus by acupuncture and then used for in vitro analysis on day 7.

CAR-NK cell production:

For CAR-NK production, cord blood (CB) units used in the study were obtained from the MD Anderson Cancer Center Cord Blood Bank. CB monocytes were isolated from frozen CB units by Ficoll density gradient centrifugation. Ex vivo expansion of cord blood-derived NK cells (CB-NK cells) was stimulated with uAPCs on day 0 in addition to IL-2 feeding every 2 days. On day 6, cells were transduced with RD114 virus using the pinprick method. A second round of uAPC-stimulated cells was added on day 8 or 9 and fed IL-2 every 2 days until day 15 for in vivo or ex vivo studies.

One day after MM1s tumor cell inoculation, 1 million or 3 million BCMA CAR-NK cells containing BCMA28-2 (approximately 70% CAR+ve) were administered to mice IV ("1-day delayed administration")

Tumor progression was monitored and imaged every 7 days. Figure 6 shows tumor growth as a function of days in mice treated with untransduced NK cells or NK cells transduced with BCMA-CAR with CD28 hinge domain in a 1-day delayed dosing regimen. Figure 7 shows 1 day delayed dose Kaplan Meier survival curves for NK cells expressing BCMA28-2. Similar experiments were repeated with a 9-day delay in administration of BCMA28-2 CAR-expressing NK cells. BCMA28-2 CAR-NK cells were observed to produce prolonged survival compared with tumor-only or non-transfected NK cells.

It was observed that switching from co-administration to delayed dosing settings (1-day and 9-day delayed dosing) continued to show positive anti-tumor effects of BCMA CAR-NK treatment.

Example 7: Secretion of IL-15 in BCMA-CAR-NK cells

This example demonstrates that NK cells transduced by the exemplary BCMA-CAR-IL-15 construct persist in vivo as measured by monitoring CAR copy number per 1000 cells and IL-15 secretion.

The detection of human in NSG mouse plasma samples using electrochemiluminescence immunoassay on the Meso Scale Discovery (MSD®) platform and the U-PLEX Human IL15 Assay Kit (Cat. No. K151URK/(BRS 20-735), supplier MSD) IL-15 bioanalysis. In the U-PLEX® assay, biotinylated capture antibodies are first coupled to streptavidin on the surface of the U-PLEX® plate. Calibrators, controls, and samples are added to the plate and incubated at room temperature with shaking. The calibrators, controls, and human IL-15 in the samples are then bound to capture antibodies. After washing, a solution containing detection antibodies conjugated to an electrochemiluminescent label (MSD GOLD SULFO-TAG) was added to the plate, followed by another incubation period. After incubation with detection antibodies, plates were washed and 2X reading buffer was added to the wells. Read the plate immediately in MSD Sector Imager S600. Robust dose-dependent expansion and increase in IL-15 production of BCMA CAR-NK cells expressing the BCMA-CAR of SEQ ID NO: 20 was observed in the delayed dosing setting, as seen in Figure 9 .

Example 8: Using the BCMA CAR construct in Improved tumor cell killing in T cell environment, etc.

This example demonstrates the use of BCMA CAR constructs (eg, BCMAIg1, BCMAIg2, BCMA28-1, BCMA28-2) in tumor cell killing activity.

Cytotoxicity in the T cell environment was assessed using Promega's CellTiter-Glow luminescent cell viability assay according to the manufacturer's protocol. To analyze apoptosis and mitochondrial damage, MM.1S-ffluc-MDA target cells were labeled with CellTracker deep red dye from Invitrogen, and the MultiCyt Apoptosis Kit from Sartorius was used to measure half of the cells according to the manufacturer's protocol. Caspase positivity and mitochondrial damage. Briefly, activation of caspases 3 and 7 is detected by using NucView 488 Caspase-3/7 substrate which fluoresces upon cleavage. Mitochondrial membrane potential is determined by the isolation of fluorescent small molecules from active membrane potential within the intact mitochondrial lumen. When mitochondrial depolarization leaks into the cytoplasm, cells exhibit a decrease in fluorescence. The iQue flow cytometer from Intellicyte is used to obtain flow cytometry Cytometer readings.

BCMA CAR constructs (e.g., BCMAIg1, BCMAIg2, BCMA28-1, BCMA28-2) were tested against the BCMA-positive cell line MM.1S-ffluc-MDA in the same CAR backbone of human primary T cells.

All BCMA CAR constructs showed greater killing than T cells expressing CD19 CAR (negative control). Furthermore, constructs utilizing the CD28 hinge showed higher levels of cytotoxicity, apoptosis, and mitochondrial damage compared to the same conjugate in the CAR backbone containing the IgG1 hinge. The specific killing of tumor cells based on three different analytical readouts is shown in Figure 10 (AC) .

Example 9: Analysis of apoptosis of transfected CAR-NK cells

This experiment was designed to analyze apoptosis in CAR-NK transduced with BCMA28-1 or BCMA28-2 in the presence or absence of soluble BCMA.

For analysis of apoptosis, MM.1S-ffluc-MDA target cells were used for CellTracker deep red dye labeling from Invitrogen, and the MultiCyt Apoptosis Kit from Sartorius was used to measure caspase positivity according to the manufacturer's protocol . Effector cells were co-cultured with target cells in the presence or absence of 800 ng/mL soluble recombinant BCMA obtained from ACRO Biosciences. Briefly, activation of caspases 3 and 7 is detected by using NucView 488 Caspase-3/7 substrate which fluoresces upon cleavage. The iQue flow cytometer from Intellicyte was used to obtain flow cytometry readings.

It was observed that in the presence of 800ng/mL soluble BCMA, especially at high E:T ratio, CAR-NK induced lower levels of apoptosis in target cells, as can be seen in Figure 11 .

Example 10: In vivo efficacy of BCMA CAR constructs against MM1S tumors

This example shows the expression of CAR-containing BCMA28-2 pairs in CB-NK cells. Efficacy of MM1S tumors.

Female NSG mice aged 10-12 weeks were irradiated whole body with 150 cGy 24 hours before tumor inoculation. MM.1S-ffluc-MDA cells were prepared in PBS suspension at a concentration of ^2.5x10 cells/ml and used to inoculate cells intravenously at 0.5x10 ^cells /animal. Bioluminescence images were taken 1 day before drug administration and 6 days after tumor inoculation, and the animals were randomly divided into groups of 4 animals per group based on the total flux. Animals were dosed 7 days after tumor inoculation. CA-expressing NK (ie, CAR NK) cells that have been cryopreserved in a frozen formulation (e.g., 38.6% PLASMA-LYTE A+50% CS10+10% HSA+0.8% AA+vitamins+30mM trehalose) are thawed and directly Inject mice with PBS buffer without washing. Bioluminescence images were performed weekly on Xenogen IVIS to monitor tumor progression. Body weights were measured three times per week, and clinical observations were performed to monitor for any signs of toxicity. Microsampling (blood collection via submandibular) was performed weekly for cell kinetic analysis to quantify in vivo CAR NK amplification by ddPCR or flow cytometric analysis. At humane or research endpoints, animals from studies of interest are necropsied to obtain various tissues for toxicological/pathological evaluation.

All CAR-transduced NK cells showed a survival benefit compared to tumor alone ( Figure 13 ).

CAR-NK cell manufacturing

For CAR-NK production, cord blood (CB) units used in the study were obtained from the MD Anderson Cancer Center Cord Blood Bank. CB monocytes were isolated from frozen CB units by Ficoll density gradient centrifugation. Ex vivo expansion of cord blood-derived NK cells (CB-NK cells) was stimulated with uAPCs on day 0 in addition to IL-2 feeding every 2 days. On day 6, cells were transduced with RD114 virus using the pinprick method. Add a second round of uAPC-stimulated cells on day 8 or 9 and feed IL-2 every 2 days until day 15 for in vivo or ex vivo studies or cryopreserve on day 21 for later use.

Example 11: In vitro efficacy of BCMA CAR constructs against various tumor strains

This example shows the efficacy of BCMA28-2 CAR expressed in CB-NK cells against different tumor cells.

MM1S-Luc, RPMI-8226-Luc, JJN3-Luc and JJN3-Luc BCMA KO cells were washed once with PBS, and the cells were mixed with a 1:10,000 dilution of cell tracer deep red dye (Invitrogen#C34565) in PBS. Incubate for 20 minutes at 37°C with a cell density of 2.5 million/ml. At the end of the incubation, add 20 ml of cell culture medium to the cells, centrifuge the cells at 500g for 5 minutes, remove the supernatant and wash the cells again with the corresponding cell culture medium. The cells were then resuspended in culture medium at 250,000/ml and 30 μl of cells were added to each well of a V-bottom 384-well assay plate (Greiner, catalog: 781280). Incubate the cells in a 37°C cell incubator with 5% CO _for 1-2 hours. Harvest fresh effector cells on day 15 and wash the cells once in interleukin-free NK cell medium (CellGenix GMP SCGM with 10% HI-FBS and 2mM glutamine), then 10 μl of effector cells were Different E:T ratios were added to the assay plate. The target cells and effector wells were co-cultured for 20 hours, then the cells were centrifuged and the supernatant was collected for interleukin release analysis. Cells were incubated with 10 μl of caspase 3/7 reagent (Intellicyt, catalog: 91035) diluted 1:500 in the corresponding target cell culture medium for 1 h at 37°C in a cell incubator before being presented for processing. Use Sartorius iQue3 or Sartorius iQue screener Plus for FACS analysis. The percentage of target cells with positive caspase 3/7 staining is used to report effector cell cytotoxicity.

In vitro killing activity was demonstrated against various tumor cell lines by NK cells containing BCMA28-2 CAR constructs derived from different CBU donors ( as shown in Figure 14A to Figure 14D ).

Example 12. The ability of BCMA CAR NK cells to damage tumor cells in vitro under multiple rounds of restimulation

This example shows the efficacy of BCMA28-2 CAR expressed in CB-NK cells.

Effector cells were harvested and resuspended in SCGM (CellGenix, catalog number: 20802-0500) supplemented with 10% heat-inactivated FBS (Sigma, catalog number: F4135-500mL), 1% L-glutamine (Gibco, catalog number: :25030-081), 1% Penn Strep (Gibco, catalog number: 15140-122), and 100IU/mL human IL-2 (Miltenyi, catalog number: 130-097-748). Effector cells were seeded in triplicate into 48-well flat-bottom non-tissue culture treated plates (Corning, catalog number: 3548) at a density of 2e5 cells/well (MM1S model). Target cells previously transduced with NucLight Red Lentivirus (Sartorius, Cat. No.: 4476) and selected with 1 μg/mL puromycin (Sigma, Cat. No.: P8833-10MG) were harvested and resuspended in the above complete medium and inoculated with 5e4 cells. / hole density inoculation. The plate was placed in an IncuCyte S3 (Sartorius Inc.) and quadruplicate readings of each well were taken every 30 minutes using a 10X objective in the brightfield and red channels. Target cells were prepared as described above and replated every 48-72 hours at a density of 5e4 cells/well (MM1S model) for a total of 9 tumor stimulations/rechallenges. Cytolysis of target cells was expressed as the average red object count per image ( Figure 15A ) and the overall average area under the red object count curve ( Figure 15B ).

Example 13: In vivo efficacy of BCMA CAR constructs against RPMI-8226 tumors

Female NSG mice aged 10-12 weeks were irradiated whole body with 150 cGy 24 hours before tumor inoculation. RPMI-8226-luc cells were prepared in PBS suspension at a concentration of ^2.5x10 cells/ml for intravenous inoculation of cells at 0.5x10 ^cells /animal. Bioluminescence images were taken 1 day before drug administration and 6 days after tumor inoculation, and the animals were randomly divided into groups of 4 animals per group based on the total flux. Animals were dosed 7 days after tumor inoculation. Relevant concentrations of CAR-expressing NK cells were resuspended in PBS and transferred to a vivarium on ice in small batches to ensure timely infusion into the animals while maintaining the viability of the CAR-expressing NK cells. Bioluminescence images were performed weekly on Xenogen IVIS to monitor tumor progression. Body weights were measured three times per week, and clinical observations were performed to monitor for any signs of toxicity. Microsampling (blood collection via submandibular) was performed weekly for cell kinetic analysis to quantify in vivo CAR NK amplification by ddPCR or flow cytometric analysis.

Compared with UTD NK, the BCMA28-2 (with CD28 costimulatory domain)-CAR NK treatment group showed anti-tumor activity ( Figure 16 ).

TW202330612A_111139693_SEQL.xml

Claims (81)

A B cell maturation antigen (BCMA) antibody or antigen-binding fragment thereof, comprising: A heavy chain variable region containing three heavy chain complementary regions (HCDR), wherein HCDR1 contains SYAIH (SEQ ID NO: 2), HCDR2 contains VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 contains AKFGEPQYFQH (SEQ ID NO: 4). The BCMA antibody or antigen-binding fragment thereof as described in claim 1, wherein the BCMA antibody or its fragment binds BCMA with a K _D greater than 0 nM and less than 150 nM. The BCMA antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the BCMA antibody or fragment thereof binds BCMA with a K _D greater than 1 pM and less than 10 nM. The BCMA antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the BCMA antibody or fragment thereof binds BCMA presented on human cells with an EC50 between 0.05 μg/ml and 0.5 μg/ml. The BCMA antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the BCMA antibody or fragment thereof comprises three light chain variable regions (LCDR), wherein the LCDR2 comprises AASTLQS (SEQ ID NO: 7) . The BCMA antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the BCMA antibody or fragment thereof comprises three light chain variable regions (LCDR), wherein the LCDR1 comprises RASQGISSYLA (SEQ ID NO: 11) , the LCDR2 contains AASTLQS (SEQ ID NO:7), and the LCDR3 contains QQLNSYPWT (SEQ ID NO:14). The BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the BCMA antibody or fragment thereof comprises three light chain variable regions (LCDR), wherein the LCDR1 comprises RASQGINNYLA (SEQ ID NO: 6), the LCDR2 contains AASTLQS (SEQ ID NO: 7), and the LCDR3 contains QQLKSYPFT (SEQ ID NO: 8). The BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the BCMA antibody or fragment thereof comprises three light chain variable regions (LCDR), wherein the LCDR1 comprises RASQGISSYLA (SEQ ID NO: 11), the LCDR2 contains AASTLQS (SEQ ID NO:7), and the LCDR3 contains QQLNSYPFT (SEQ ID NO:12). The BCMA antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the BCMA antibody or fragment thereof is an antibody comprising an IgG constant region. A B cell maturation antigen (BCMA) antibody or antigen-binding fragment thereof, comprising three LCDRs, wherein, LCDR1 contains RASQGIX ₁ X ₂ YLA (SEQ ID NO: 79), LCDR2 contains AASTLQS (SEQ ID NO: 7), and/or LCDR3 contains QQLX ₃ SYPX ₄ T (SEQ ID NO: 80); Among them, X ₁ is selected from S or N; X ₂ is selected from S or N; X ₃ is selected from N or K; and/or X ₄ is selected from F or W. The BCMA antibody according to any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a heavy chain variable region (VH), the VH comprising SEQ ID NO: 9. The BCMA antibody according to any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a VH sequence that is at least 95% identical to SEQ ID NO: 9. The BCMA antibody according to any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a heavy chain variable region (VH), the VH comprising SEQ ID NO: 1. The BCMA antibody according to any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a VH sequence that is at least 95% identical to SEQ ID NO: 1. The BCMA antibody of any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a light chain variable region (VL), the VL comprising SEQ ID NO: 5, 10 or 13. The BCMA antibody according to any one of the preceding claims, wherein the antibody or the antigen-binding fragment comprises a VL sequence that is at least 90% identical to SEQ ID NO: 5, 10 or 13. The anti-BCMA antibody or antigen-binding fragment thereof as described in any one of the preceding claims, wherein the anti-BCMA antibody or fragment thereof is selected from the group consisting of: IgA antibody, IgG antibody, IgE antibody, IgM antibody, bi- or multispecific antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or groups thereof; single chain variable fragments (scFv), polypeptide-Fc fusions, Single-domain antibodies, camel antibodies; masking antibodies, Small Modular ImmunoPharmaceutical ("SMIPsTM"), single-chain, tandem bivalent antibodies, VHH, anticarbalin, nanobodies, microbodies, BiTE, anchors Protein repeat protein, DARPIN, Avimer, DART, TCR-like antibody, Adenastin, human ubiquitin, transmembrane antibody (Trans-body); Affibody, TrimerX, MicroProtein, Fynomer, Centyrin; and KALBITOR. The BCMA antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the BCMA antibody or fragment thereof is a single chain variable fragment (scFv). The BCMA antibody or antigen-binding fragment thereof according to claim 18, wherein the BCMA-binding scFv includes a linker sequence. The BCMA antibody or antigen-binding fragment thereof according to claim 19, wherein the BCMA-binding scFv comprises a linker selected from SEQ ID NO: 15-18. The BCMA antibody or antigen-binding fragment thereof according to claim 18, wherein the BCMA-binding scFv includes a signal peptide. The BCMA antibody or antigen-binding fragment thereof as described in claim 18, wherein the BCMA-binding scFv comprises a sequence selected from the group consisting of SEQ ID NOs: 85-87. The BCMA antibody or antigen-binding fragment thereof according to claim 22, wherein the BCMA-binding scFv comprises a sequence that is at least 80% identical to SEQ ID NO: 85, 86 or 87. A BCMA antibody or antigen-binding fragment that competes with an antibody or antigen-binding fragment comprising: A heavy chain variable region containing three heavy chain complementary regions (HCDR), wherein HCDR1 contains SYAIH (SEQ ID NO: 2), HCDR2 contains VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 contains AKFGEPQYFQH (SEQ ID NO: 4). A BCMA antibody or antigen-binding fragment that competes with an antibody or antigen-binding fragment containing three LCDRs, wherein, LCDR1 contains RASQGIX ₁ X ₂ YLA (SEQ ID NO: 79), LCDR2 contains AASTLQS (SEQ ID NO: 7), and/or LCDR3 contains QQLX ₃ SYPX ₄ T (SEQ ID NO: 80); Among them, X ₁ is selected from S or N; X ₂ is selected from S or N; X ₃ is selected from N or K; and/or X ₄ is selected from F or W. A method of treating cancer, the method comprising administering to an individual in need of treatment a BCMA antibody or an antigen-binding fragment thereof as described in any one of the preceding claims. The method of claim 26, wherein the cancer is an immune cell malignancy, such as leukemia, lymphoma or myeloma. A pharmaceutical composition comprising a BCMA antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, wherein the BCMA antibody or an antigen-binding fragment thereof contains: A heavy chain variable region containing three heavy chain complementary regions (HCDR), wherein HCDR1 contains SYAIH (SEQ ID NO: 2), HCDR2 contains VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 contains AKFGEPQYFQH (SEQ ID NO: 4). A pharmaceutical composition comprising a BCMA antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, wherein the BCMA antibody or fragment thereof contains: Light chain variable complementarity determining region (LCDR) 2 comprising AASTLQS (SEQ ID NO: 7). A polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antigen-binding molecule that specifically binds to B cell maturation antigen (BCMA), a CD28 hinge region, a transmembrane domain, and one or more intracellular cell signaling transducers area. The polynucleotide of claim 30, wherein the antigen-binding molecule that specifically binds BCMA includes: (a) Heavy chain variable region (VH) complementarity determining region (CDR) 1, the VH CDR1 comprising SEQ ID NO: 2; (b) VH CDR2, the VH CDR2 comprising SEQ ID NO: 3; and (c) VH CDR3 comprising SEQ ID NO:4. The polynucleotide of claim 31, wherein the antigen-binding molecule that specifically binds BCMA includes: (a) Light chain variable region (VL) complementarity determining region (CDR) 1, the VL CDR1 comprising SEQ ID NO: 6; (b) VL CDR2 comprising SEQ ID NO: 7; and (c) VL CDR3 comprising SEQ ID NO:8. The polynucleotide of claim 32, wherein the antigen-binding molecule that specifically binds BCMA includes: (a) Light chain variable region (VL) complementarity determining region (CDR) 1, the VL CDR1 comprising SEQ ID NO: 11, (b) VL CDR2 comprising SEQ ID NO: 7, and (c) VL CDR3 comprising SEQ ID NO:12. The polynucleotide of claim 31, wherein the antigen-binding molecule that specifically binds BCMA includes: (a) Light chain variable region (VL) complementarity determining region (CDR) 1, the VL CDR1 comprising SEQ ID NO: 11, (b) VL CDR2 comprising SEQ ID NO: 7, and (c) VL CDR3 comprising SEQ ID NO:14. The polynucleotide of any one of claims 30 to 34, wherein the antigen-binding molecule that specifically binds BCMA comprises at least about 95%, at least about 96%, at least about 97% of SEQ ID NO: 1 or 9 , an amino acid sequence that is at least about 98%, at least about 99%, or about 100% identical. The polynucleotide of any one of claims 30 to 35, wherein the antigen-binding molecule that specifically binds BCMA comprises at least about 95%, at least about An amino acid sequence that is 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical. The polynucleotide of any one of the preceding claims, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, F(ab')2 fragment, Fv fragment or single-chain variable fragment (scFv). The polynucleotide of any one of claims 30 to 37, wherein the antigen-binding molecule comprises scFv. The polynucleotide of any one of claims 30 to 38, wherein the VH and the VL are connected by a linker. The polynucleotide of claim 39, wherein the linker contains about 50 amino acids to about 2 amino acids. The polynucleotide of claim 39, wherein the linker comprises at least 75%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 15-18 %, at least 98%, at least 99% or 100% identical amino acid sequences. The polynucleotide of any one of claims 30 to 41, wherein the antigen-binding molecule is less than about 1×10 ^-6 M, less than about 1×10 ^-7 M, less than about 1×10 ^-8 M or Binds BCMA with a K _D less than about 1×10 ^-9 M. The polynucleotide of any one of claims 30 to 42, wherein the transmembrane domain is the following transmembrane domain: CD28, CD3ζ, CD3 ε, CD3 γ, CD3 δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, or any combination thereof. The polynucleotide of claim 43, wherein the transmembrane domain is the CD28 transmembrane domain. The polynucleotide of any one of claims 30 to 44, wherein the hinge region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about SEQ ID NO: 56 An amino acid sequence that is 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical. The polynucleotide of any one of claims 30 to 45, further comprising a costimulatory region. The polynucleotide of claim 46, wherein the costimulatory region has the following signal Transduction zone: CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), CD8 γ, CD3 δ, CD3 ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF1.4), NKG2C, Ig α (CD79a), Fc γ receptor, MHC class I molecule, TNF receptor Body proteins, immunoglobulin-like proteins, interleukin receptors, integrins, signaling lymphoid activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7 -H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8α, CD8β, 11.2 β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, TNFR2, TRANCE RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9(CD229), CD160(BY55), PSGL1, CDIOO(SEMA4D) , CD69, SLAMF6 (NTB-A, Lyl08), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD 19a, ligands that specifically bind to CD83 or any of them combination. The polynucleotide of any one of claims 30 to 47, wherein the costimulatory region comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least SEQ ID NO: 28 An amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical. The polynucleotide of any one of claims 30 to 48, further comprising an activation domain. The polynucleotide of claim 49, wherein the activation domain is a CD3ζ domain. The polynucleotide of claim 49 or 50, wherein the activation domain comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least SEQ ID NO: 30 About 7. An amino acid sequence that is at least about 98%, at least about 99%, or about 100% identical. The polynucleotide according to any one of claims 30 to 51, further comprising a suicide gene. The polynucleotide of claim 52, wherein the suicide gene is selected from the group consisting of rituximab, iCaspase 9, herpes simplex virus-thymidine kinase (HSV-tk), and ganciclovir, acyclovir or FIAU ;Oxidoreductase and cycloheximide; Cytosine deaminase and 5-fluorocytosine; Thymidine kinase thymidylate kinase (Tdk::Tmk). The polynucleotide of any one of claims 52 to 53, wherein the suicide gene is iCaspase9. The polynucleotide of any one of claims 30 to 54, further comprising an interleukin. The polynucleotide of claim 55, wherein the interleukin is selected from IL-7, IL-12, IL-15, IL-18 or IL-21. The polynucleotide of any one of claims 55 to 56, wherein the interleukin is IL-15. The polynucleotide of claim 57, wherein the amino acid sequence of IL-15 includes SEQ ID NO: 23. The polynucleotide of any one of claims 30 to 58, wherein the CAR comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least SEQ ID NO: 19-21 An amino acid sequence that is about 96%, at least about 7, at least about 98%, at least about 99%, or about 100% identical. A vector comprising the polynucleotide of claim 59. The vector as described in claim 60 is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, an adenovirus-related vector, a lentiviral vector or any combination thereof. A CAR encoded by the polynucleotide of any one of claims 30 to 59. A cell comprising the polynucleotide of any one of claims 30 to 59, the vector of any one of claims 60 to 61, the CAR of claim 62, or any combination thereof. The cell of claim 63, wherein the cell is an immune cell. The cell according to any one of claims 63 to 64, wherein the cell is a NK cell, a T cell or a tumor-infiltrating lymphocyte (TIL), an iNKT cell, a B cell, a macrophage, a dendritic cell or a mixture thereof. A composition comprising the polynucleotide as described in any one of claims 30 to 59, the vector as described in any one of claims 60 to 61, the CAR as described in claim 62, or the CAR as claimed in claim 62 The cell described in any one of 63 to 65. An immune cell population comprising cells according to any one of claims 63 to 66. A method of treating cancer in an individual comprising the step of administering to the individual a therapeutically effective amount of the composition of claim 66. The method of claim 68, further comprising the step of providing an effective amount of additional therapy to the individual. The method of claim 68, wherein the additional therapy includes surgery, radiation, gene therapy, immunotherapy or hormonal therapy. The method according to any one of claims 68 to 70, wherein by infusion, injection, intravenous, intraarterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intralesional, intracranial , administering cells containing the polynucleotide or cells containing the vector to an individual transdermally, subcutaneously, topically, by perfusion, in the tumor microenvironment, or a combination thereof. The method of any one of claims 68 to 71, wherein the cancer is selected from multiple myeloma, lymphoma and/or leukemia. An NK cell comprising a polynucleotide encoding a CAR, the CAR comprising: (a) An antigen-binding molecule that specifically binds to BCMA, comprising A heavy chain variable region (VH) that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1 or 9, and A light chain variable region (VL) that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% consistent with any one of SEQ ID NO: 5, 10, or 13 or about 100% agreement; (b) CD28 hinge region; (c) Transmembrane domain; and (d) One or more intracellular cell signaling domains. A polynucleotide encoding a CAR that binds BCMA, comprising: (a) CD28 hinge, (b) transmembrane domain, (c) costimulatory domain, and (d) IL-15 interleukin. An NK cell comprising the polynucleotide described in claim 74. An immune cell having a polynucleotide encoding a chimeric antigen receptor (CAR), which The CAR contains: (a) Antigen binding domain, (b) CD28 hinge, and (c) CD28 transmembrane domain. The immune cell of claim 76, wherein the cell comprises a CAR that binds to BCMA expressed on tumor cells. A polynucleotide comprising a nucleotide sequence encoding an anti-BCMA antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 25. The polynucleotide of claim 78, comprising at least one chemical modification. The polynucleotide of claim 78, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: SEQ ID Nos: 55-56, 58-59, 63, 67, 71 and 76-78 . An NK cell expressing a CAR as described in claim 62 that binds BCMA.

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